Smart modelling of unusual cyanobacteria – an enigma solved?

Gloeothece is an unusual cyanobacterium because it is capable of fixing atmospheric N2 aerobically, even though nitrogenase is only active in the absence of O2 (Fig. 1) (Gallon, 1992). But how do we explain this enigma? That question is addressed in this issue of New Phytologist (pp. 545–555), in a paper from Nicholas Stephens, Kevin Flynn and, posthumously, John Gallon that brings together more than 30 years of research on this organism. It is an impressive account of what we know, integrated in a smart mathematical model. Is the enigma solved? Micrograph of Gloeothece PCC 6909. Globally, N2 fixation is the most important process after CO2 fixation. It is the only process that counteracts the losses of ‘bound’ nitrogen, NH4+ and NO3−, through denitrification and anaerobic ammonium oxidation (anammox), which convert it to N2. However, although it is the most abundant form of nitrogen on earth, N2 can only be used by a few specialized, prokaryotic (Bacteria and Archaea) microorganisms. This is a remarkable achievement if one realizes that the triple bond between the two N atoms is very strong and the chemical reduction of N2 to NH3 is possible only at very high temperature (400–650°C) and pressure (200–400 atmosphere) (Haber-Bosch process). Although diazotrophic microorganisms can perform N2 fixation at ambient temperature and pressure, it is nevertheless at the expense of much energy and low-redox potential electrons. From this perspective it does not come as a surprise that many cyanobacteria possess the genetic potential to fix N2. They are the only diazotrophic primary producers, combining the fixation of N2 and CO2. The O2-evolving photosynthetic cyanobacteria had to find ways to accommodate nitrogenase, which is incompatible with the presence of O2. In 1968 Peter Fay et al. discovered that the heterocyst is the site of N2 fixation (Fay et al., 1968). Until then it was known that filamentous, heterocystous cyanobacteria were capable of N2 fixation, but the function of the differentiated cells, the heterocysts, was unknown. A few years later Donze et al. (1972) demonstrated that the heterocyst did not possess photosystem II and therefore would be incapable of evolving O2 or fixing CO2. It was not until the 1980s that Anthony Walsby proved that the heterocyst was essentially anoxic (Walsby, 1985) and, finally, that the group of Birgitta Bergman demonstrated the presence of nitrogenase antigen in the heterocyst using immunology (Braun-Howland et al., 1988). The plethora of evidence for the heterocyst as the explanation for the presence of the incompatible processes of oxygenic photosynthesis and N2 fixation in one organism was rudely interfered with by the publication, only a year after Fay's paper in Nature, of a report in Science that unequivocally demonstrated the capability of the unicellular cyanobacterium Gloeocapsa (later re-assessed as Gloeothece) to grow diazotrophically (Wyatt & Silvey, 1969). John Gallon decided to solve this puzzle, starting to work with W. G. W. Kurz and T. A. La Rue in Canada in the early 1970s until his early death this year. The model that is presented in the paper of Stephens et al. relies to a large extent on the results obtained by the group of John Gallon since. The model simulates the various features that have been observed in Gloeothece experimentally. One of these features is the fact that N2 fixation is temporarily separated from photosynthesis when the organism is grown under an alternating light/dark cycle or in its natural environment under ambient conditions. Both processes are spatially separated in heterocystous cyanobacteria, while temporal separation has been observed in most other filamentous and unicellular non-heterocystous cyanobacteria (Gallon & Stal, 1992; Bergman et al., 1997). However, there is an important exception to this rule. Filamentous non-heterocystous Trichodesmium spp., which occur in the pelagic tropical oceans, exhibit a behaviour very similar to that of heterocystous species – N2 fixation is confined to the daytime. Nevertheless, a small but important difference is that all heterocystous cyanobacteria also fix N2 during the night, although at a significantly lower rate (typically one third of the rate at saturating light), while N2 fixation in Trichodesmium ceases at night and nitrogenase is even inactivated and degraded (Zehr et al., 1993). There is compelling evidence that this cyanobacterium also exhibits a form of cell differentiation (Lundgren et al., 2001). Diazocytes seem to possess many of the characteristics of a heterocyst, albeit without the typical morphological features. The lack of the thick glycolipid cell envelope of diazocytes should be noted. Also, diazocytes occur in chains, while heterocysts always occur as single cells between vegetative cells or as terminal cells. Trichodesmium spp. are considered to be globally important N2 fixers (Capone et al., 1997). Currently, it is estimated that these organisms may contribute c. 50% of global biologically fixed N2 (excluding the anthropogenic contribution) (Karl et al., 2002). The contribution of Gloeothece to the global N cycle is vanishingly low, but as a model organism it is of great importance. If this model does have the potential to be adapted to describe N2 fixation in heterocystous cyanobacteria and in Trichodesmium, as claimed by the authors, it would represent a breakthrough. Such a model would not only have the potential to predict N2 fixation in the world's oceans, but it would also help us to understand CO2 fixation and oceanic primary productivity. Equally important would be the possibility of modelling N metabolism and N2 fixation in heterocystous cyanobacteria. These organisms are known to produce extensive blooms in freshwater lakes and brackish environments and many of them are toxic. For instance, the every summer the Baltic Sea suffers from extensive surface blooms of the heterocystous cyanobacteria Nodularia and Aphanizomenon (Stal & Walsby, 2000). While the former produces the hepatotoxin nodularin, the latter is nontoxic. This emphasizes the importance of understanding their different physiologies and using models to predict the success of one or the other organism. Last year, John Gallon and colleagues published a paper showing the processes during the initiation of a bloom (Gallon et al., 2002). The model of Gloeothece was built on the ammonium–nitrate interaction model (ANIM) of Flynn et al. (1997), extended with components to describe photoacclimation and N2 fixation. The authors aimed at using the essential biochemical interactions in the organism rather than empirical curve fitting. The model should be able to simulate the behaviour of Gloeothece, and in Table 4 of their paper the authors summarise the aspects of N2 fixation behaviour displayed by this organism and which must be accommodated. The model is based on the fact that any organism, when available, would have a preference for ammonium as the reduced form of nitrogen, which can immediately be assimilated into amino acids and is therefore ‘cheaper’ than nitrate. As is the case with ammonium, nitrate must be actively transported into the cell and it has to be reduced to ammonium before it can be incorporated. Although the cost of nitrate reduction is considerable, it is still far less than the cost of N2 fixation. The fixation of N2 requires 16 ATP and 8 low-redox potential electrons, in the form of reduced ferredoxin. The model simulations clearly show the use of the different nitrogen sources in order of preference. A central role in the model is given to the cellular N : C ratio, which represents the general nitrogen status. A high ratio would indicate nitrogen sufficiency and a low availability of storage carbon, which is glucan in Gloeothece, and triggers the fixation of carbon in the light. This subsequently causes the N : C ratio to decrease, moving the cell into the direction of nitrogen depletion and a high availability of reductant. This controls N2 fixation, while nitrogenase activity is subject to turn-over and inhibition by O2. Reserve glucan is respired, resulting in the production of ATP as well as the lowering of O2. Hence, N2 fixation depends on the catabolism of carbon reserves and is only indirectly supported by photosynthesis. This makes sense since nitrogenase activity in Gloeothece is confined to the dark phase when grown under alternating light/dark cycles. However, Gloeothece is also capable of diazotrophic growth under continuous light. From experiments it was demonstrated that nitrogenase activity exhibits a cycle with a period of c. 40 h. The model could simulate such behaviour only when the growth rate was lowered. This is also in agreement with experimental evidence that shows that growth under continuous light is poorer. This is probably the result of decreased nitrogenase activities that would lead to lower growth rates. This was further substantiated by Stephens et al. by running the model at low light irradiance. When provided at a 12 h/12 h light/dark cycle, nitrogenase peaked only every second dark period. It is unclear whether the model is able to mimic the behaviour of Gloeothece at higher growth rates than those that are usually achieved in batch culture. Ortega & Stal (1991) grew Gloeothece PCC 6909 in continuous culture at various light regimes and dilution rates. With a 12 h/12 h light/dark regime they grew the organism at a dilution rate of 0.007 h−1 (growth rate 0.17 d−1, estimated maximum growth rate c. 0.2 d−1), while at 16 h/8 h light/dark a maximum growth rate was c. 0.5 d−1. These growth rates are substantially higher than those that can be obtained in batch culture and which the model of Stephens et al. used. However, it was conspicuous that these cultures confined most of the N2 fixation to the light period and that the activities achieved in the light were three orders of magnitude higher than those from batch cultures, which fixed N2 exclusively in the dark (Fig. 2). The high nitrogenase activities observed in the continuous cultures were in agreement with what was expected to accommodate the imposed growth rate. This pattern is similar to that observed in heterocystous cyanobacteria and in Trichodesmium. Nevertheless, in Gloeothece the unexpected behaviour of N2 fixation in the light, presumably simultaneously with oxygenic photosynthesis, remains unexplained. When the model can be adapted to show this particular behaviour, it may become an important instrument to explain and understand N2 fixation in this organism, and – eventually – solve the enigma. Patterns of nitrogenase activity (acetylene reduction) in cultures of Gloeothece PCC 6909 grown under 12 h/12 h light/dark cycle. (a) Batch culture. (b) Continuous culture grown at D = 0.007 h−1. Note the difference in scale which is three orders of magnitude smaller in panel a (nmol) compared with panel b (µmol). (Stal, 1995).

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