In the free-living state, heterocyst differentiation is repressed by the presence of a source of combined nitrogen. When the combined nitrogen is exhausted, heterocysts appear in the filaments in a nonrandom spacing pattern, typically as a single heterocyst flanked by 10 to 15 vegetative cells (Fig. 1B). There are two interrelated phenomena with respect to the presence and position of heterocysts in a filament. The first is the sequence of heterocyst appearance following exhaustion of combined nitrogen. Based on morphological observations and mutant analyses, the developmental sequence can be generally described in three stages: (i) initiation, (ii) transition and (iii) commitment.
Initiation. In the initiation stage no distinct morphological events are manifest. The vegetative cells of mutants arrested at this stage show no signs of differentiation and the cultures eventually die in the absence of NH4+. Genes encoding proteins we have classified as positive regulatory elements (Table 4), because mutants do not initiate heterocyst differentiation, are involved at some point in this stage (Meeks and Elhai 2002). These currently include ntcA, hanA, hetR, hetF and patA, but there are likely to be additional elements. NtcA senses the nitrogen limitation signal and activates genes involved in acquisition of nitrogen sources alternative to NH4+ (Herrero et al. 2001, 2004), including hetR transcriptional enhancement. HanA is a DNA binding protein with multiple cellular roles, not specifically heterocyst differentiation (Khudyakov and Wolk 1996). HetR is considered the primary positive element in the induction of heterocyst differentiation (Buikema and Haselkorn 2001; Huang et al. 2004). HetR enhances its own transcription (Black et al. 1993) and that of ntcA (Herrero et al. 2004), and is localized in the differentiating cells (Black et al. 1993; Wong and Meeks 2001). HetF is required for HetR autoinduced transcriptional enhancement and localization of HetR to developing heterocysts (Wong and Meeks 2001). PatA is required for intercalary vegetative cell differentiation into heterocysts; the mutant forms heterocysts only at the ends of the filaments (Liang et al. 1992). When overexpressed in trans, patS, and an exogenously supplied C-terminal pentapeptide of PatS, represses the initiation of heterocyst differentiation (Yoon and Golden 1998, 2001). These are the characteristics of a negative regulatory element.
Transition. Transition to the proheterocyst stage is accompanied by the degradation of photosynthetic pigments in specific cells and an enlargement of the same cell. Mutants cells arrested in the proheterocyst stage do not synthesize nitrogenase, but a pattern of nonfluorescent cells that are spaced in the filaments similar to the final pattern of heterocyst spacing can be detected. Proheterocysts will regress to a vegetative cell state if the culture is supplied with a source of combined nitrogen (Meeks and Elhai 2002). Candidate genes encoding proteins necessary for continued development of proheterocysts are hetC (Khudyakov and Wolk 1997) and hetP (Fernandez-Pinas et al. 1994). Commitment. Commitment of proheterocysts to terminal differentiation of mature heterocysts is accompanied by deposition of the unique polysaccharide and glycolipid envelope, decreased cellular O2 tension, and induction of genes for synthesis, assembly and function of nitrogenase. There is growing list of genes encoding proteins essential for heterocyst function in an oxic environment (Herrero et al. 2004; Meeks 2005).
The second phenomenon relates to the spacing pattern. Three separate processes may be considered with regard to the spaced pattern of heterocysts in a filament: establishment, maintenance, and disruption of pattern. These processes and the initiation of differentiation are depicted in the schematic in Fig. 6.
Nitrogen limitation leading to: (A) biased initiation ol differentiation, resolution and establishment of pattern, followed by (B) maintenance of pattern or alternatively (C) symbiotic disruption of pattern
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initiation of differentiation in non randomly spaced clusters of cells
resolution of unstable clusters to no or a single differentiating cell ooocooo«oocoooocccoooo»ooocooooxco maturation of heterocysts and growth of vegetative cells
maturation of heterocysts and growth of vegetative cells differentiation of new heterocysts in the interval differentiation of new heterocysts in the interval ooococo«occcaxoooooocnxccootocoœ
signals From symbiotic plant partner leading to multiple singular heterocysts with high nitrogen fixation
signals From symbiotic plant partner leading to multiple singular heterocysts with high nitrogen fixation multiple contiguous heterocysts with low nitrogen fixation
Fig. 6. Schematic of the sequence of events and major regulatory elements participating in: A Initiation of heterocyst differentiation and establishment of the pattern of spacing; B maintenance of the pattern; and C symbiotic disruption of the pattern multiple contiguous heterocysts with low nitrogen fixation
Fig. 6. Schematic of the sequence of events and major regulatory elements participating in: A Initiation of heterocyst differentiation and establishment of the pattern of spacing; B maintenance of the pattern; and C symbiotic disruption of the pattern
Establishment. Establishment of pattern involves those gene products that are required for initiation of the differentiation cascade as noted above. The factors involved in determining specifically which cells will initiate differentiation are currently unknown; we have speculated that position in the cell cycle when the environmental signal to differentiate is received is crucial (Meeks and Elhai 2002). Overexpression of the positive acting factors HetR and HetF results in clusters of heterocysts at single sites in the filaments (multiple contiguous heterocysts; MCH). Inactivation of four of the known negative regulatory elements also results in a MCH pattern. These mutant traits support the idea that initiation of differentiation occurs in a cluster of susceptible cells (biased initiation). The model for establishment of pattern holds that the cluster of differentiating cells are in an unstable developmental state and the interaction between positive and negative regulatory elements determines whether the cells will regress or continue to differentiate (resolution) (Meeks and Elhai 2002). HetR and PatS are projected to be the dominant positive and negative regulatory elements, respectively, with other elements acting in an ancillary role.
Maintenance. The spacing pattern is maintained as the vegetative cells grow and divide, using fixed nitrogen provided by the heterocysts. New heterocysts emerge at points approximately midway in the expanding vegetative cell interval between adjacent existing heterocysts. A distinct maintenance mechanism is implied by the observation that the patS mutant MCH phenotypic approaches the wild-type spacing pattern of single heterocysts at a site as the culture grows and new heterocysts differentiate (Yoon and Golden 2001). The negative regulatory elements HetN (Callahan and Buikema 2001) and PatB (Jones et al. 2003) appear to be involved more in maintenance than in establishment of the pattern. Controlled expression of hetN showed that the MCH phenotype of the mutant appeared only after the initial round of heterocyst differentiation following nitrogen deprivation had occurred (i.e., second generation heterocyst differentiation), paralleling the culture time when PatS apparently no longer influences the pattern.
Disruption. The pattern can be disrupted by certain chemical treatments (Meeks and Elhai 2002), in specific mutants (Table 4), and naturally by symbiotic association with a photosynthetic eukaryotic partner (see below). All chemical treatments and all but one of the mutations result in the differentiation of multiple heterocysts at a single site. The patN mutant yields multiple heterocysts, but they are present at single sites in the filaments with a decreased number of vegetative cells in the interval between heterocysts (termed multiple singular heterocysts; MSH) (Meeks et al. 2002). Symbiotic heterocyst frequencies in plant tissues range from 15 to 60% of the total cells and both MCH and MSH patterns can be observed (Adams 2000; Meeks and Elhai 2002; Rai et al. 2002).
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