D. CONTROL OF PHOTOSYNTHESIS IN CYANOTHECE SP. ATCC 51142
Cyanothece sp. ATCC 51142 is a marine, unicellular cyanobacterium capable of N2 fixation. Because nitrogenase (the enzyme which fixes atmospheric N2) is highly sensitive to oxygen, cells use a variety of strategies to limit the amount of intracellular O2. We have found that Cyanothece uses a form of temporal regulation, in that N2 fixation and photosynthesis each occur at 24-hour intervals and 12 hours out of phase. These metabolic periodicities are true circadian rhythms and there are now other examples of circadian rhythms in prokaryotes (Huang, et al., 1993; Kondo et al., 1993). These rhythms are enormously stable and can persist for weeks. Indeed, we have determined that there is a marvelous interplay among all the major metabolic processes, including respiration, carbohydrate biosynthesis and storage and nitrogen storage and signaling (in the form of the compound cyanophycin). However, the key to the entire process is the way in which PSII and O2 evolution are controlled throughout the diurnal cycle. We have found similarities in Cyanothece to various parameters that we have detected in Synechocystis mutants. Thus, Cyanothece provides an excellent opportunity to analyze dynamic changes in PSII under true physiological conditions (Reddy et al., 1993; Schneegurt et al., 1994, 1997a, b; Colón-López et al., 1997).
We first found that, at times in the daily cycle, Cyanothece resembles the Synechocystis DpsbO mutant. The periodicities mentioned above can be seen whether cells are grown under 12 hour light/12 hour dark (LD), or continuous light (LL) or continuous dark (DD) conditions. O2 evolution reaches a peak somewhere around 6-8 hours in the light and then declines well before cells go into the dark growth phase. We first discovered some interesting phenomena in Cyanothece by providing the typical dark incubation prior to measuring O2-flash yields. We found that as little as 5 minutes of dark adaptation led to O2 yields that were very small and that lack the typical period for oscillation. These cells could then be reactivated by a few minutes of continuous flashing light. On the other hand, Cyanothece cells that were grown in the dark showed a rather higher level of stability. The relationship to similar properties in the Synechocystis DpsbO mutant allows us to hypothesize that Cyanothece PSII is controlled, in part, by the extrinsic lumenal proteins of the OEC and one aspect of this control leads to instability of the Mn complex at certain times of the growth cycle. Thus, Cyanothece provides an excellent model system for analysis of dynamic regulation in PSII. A diagram of the regulation of transcriptional and photosynthetic events is presented in Fig. 4.
1. Photoactivation Cycles of PSII in Cyanothece sp. ATCC 51142The analysis of O2-flash yields in Cyanothece sp. ATCC 51142 indicated a substantial level of PSII heterogeneity at times in the diurnal cycle. The changes were complex and involved many different aspects of PSII structure and function. We obtained evidence of changes on both the oxidizing and reducing sides of PSII; these changes need to be related to changes in the structure of PSII centers, the organization of the photosystems and the location of electron transport components (Meunier et al. 1997a, 1998a; Sherman et al., 1998).
We determined a substantial amount of heterogeneity for O2 evolution in PSII at different times in the Cyanothece sp. ATCC 51142 cycle. Overall, there are three different types of considerations in understanding PSII during the Cyanothece sp. ATCC 51142 life cycle: 1) stability--dark stability vs. inactivation of PSII centers; 2) photoactivation--slow (newly formed PSII centers) vs. fast and reversible. There is also the extent of photoactivation, which is based on the amount of PSII centers available at a time. This level changes in the dark relative to the light; 3) PSII heterogeneity--this relates active vs. inactive centers, but also takes into account efficient centers vs. those that have been downregulated and appear to be unstable and inefficient. In other work we have done involving gene regulation studies (Colón-López et al., 1997, 1998), we know that biosynthetic changes in PSII occur, but that these changes are relatively minor relative to the short-term regulatory changes that lead to the above heterogeneity. This can be seen rather dramatically based on the information below.
2. State Transitions and Photosystem Oligomerization AlterationsThe PSII heterogeneity discussed above can be due to either short-term or long-term regulation. The long term is based on gene regulation and reflects changes in transcription and translation patterns of specific genes and proteins. The short term is based on reorganization of existing components and can be monitored in cyanobacteria by measuring features such as state transitions. State transitions were first detected in cyanobacteria by Murata (1969), who noted the effect of light quality on the relative activity of PSI and PSII. Preferential excitation of PSI caused an increase in energy transfer to PSII and a small decrease in energy transfer to PSI (state 1), whereas preferential excitation of PSII or incubation in darkness reversed this effect (state 2). A model for state transitions in cyanobacteria has been developed by Rögner and colleagues (Rögner et al., 1996, Bald et al., 1996), who have also invoked the oligomeric state of PSI and PSII in the overall mechanism. In this model, state 1 (which favors linear electron flow from O2 production to CO2 fixation) has a dimeric PSII and monomeric PSI. Under these circumstances, the phycobilisomes are primarily attached to PSII. State 2 (which favors cyclic electron flow) had trimeric PSI complexes and monomeric PSII. In this case, the phycobilisomes could more readily attach to PSI. The relationship of oligomerization of the photosystems and state transitions have been an important area of study. We have seen PAM and 77K fluorescence patterns which change virtually hourly throughout the diurnal cycle in Cyanothece sp. ATCC 51142.
The key finding in these experiments was the significant state transitions that occur throughout the Cyanothece sp. ATCC 51142 diurnal cycle. The fundamental properties are described in Meunier et al., 1997a and 1998a. The first paper describes the changes in 77K fluorescence as a function of growth time. Utilizing phycobilisome excitation, it¹s clear that there is a greater percentage of state 1 as we go through the light period and a significant jump to state 1 at a time immediately after N2 fixation during dark growth (D6).
These results led us to investigate the interaction of redox control and state transitions in Cyanothece. Much of this work is reviewed and analyzed in the mini-review published in Photosynthesis Research (Sherman, et al., 1998). Cyanothece represents an excellent physiological system to analyze these changes because of the significant shifts between respiration and photosynthesis. Thus, there are major changes in the PQ redox pool under physiological conditions, a system which does not require utilizing artificial conditions such as high light or photosynthetic inhibitors.
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