c-di-GMP Homeostasis Is Critical for Heterocyst Development in Anabaena sp. PCC 7120

c-di-GMP is a ubiquitous bacterial signal regulating various physiological process. Anabaena PCC 7120 (Anabaena) is a filamentous cyanobacterium able to form regularly-spaced heterocysts for nitrogen fixation, in response to combined-nitrogen deprivation in 24h. Anabaena possesses 16 genes encoding proteins for c-di-GMP metabolism, and their functions are poorly characterized, except all2874 (cdgS) whose deletion causes a decrease in heterocyst frequency 48h after nitrogen starvation. We demonstrated here that c-di-GMP levels increased significantly in Anabaena after combined-nitrogen starvation. By inactivating each of the 16 genes, we found that the deletion of all1175 (cdgSH) led to an increase of heterocyst frequency 24h after nitrogen stepdown. A double mutant ΔcdgSHΔcdgS had an additive effect over the single mutants in regulating heterocyst frequency, indicating that the two genes acted at different time points for heterocyst spacing. Biochemical and genetic data further showed that the functions of CdgSH and CdgS in the setup or maintenance of heterocyst frequency depended on their opposing effects on the intracellular levels of c-di-GMP. Finally, we demonstrated that heterocyst differentiation was completely inhibited when c-di-GMP levels became too high or too low. Together, these results indicate that the homeostasis of c-di-GMP level is important for heterocyst differentiation in Anabaena.

Effects of PatU3 peptides on cell size and heterocyst frequency of Anabaena sp. PCC 7120

patU, one of the genes specifically found in filamentous cyanobacteria, is required for the pattern formation in heterocyst-forming species. In Anabaena sp. PCC 7120, patU is split into patU5 and patU3, and only patU3 is involved in heterocyst patterning. Here, we report that PatU3 is also involved in control of cell size. A patU3 deletion mutant showed remarkably smaller cell size and much higher heterocyst frequency than that of the wild type. Yeast two-hybrid and pull-down assays demonstrated a direct interaction between PatU3 and the cell division protein Ftn6. Without the N-terminal 16-aa portion (MQERFQAVIKRRLQIH), PatU3 was no longer able to interact with Ftn6. This portion of PatU3 is also required for the interaction with PatN, a protein related to heterocyst differentiation/patterning. Addition of the 16-aa peptide or AVIKRRLQ-containing peptides restored the cell size and heterocyst frequency of a patU3 deletion mutant to normal or near wild-type level. PatU3(1-16aa)-GFP, the N-terminal 16-aa sequence fused with GFP, formed polar aggregates and peripheral patches in heterocysts of Anabaena 7120, whereas PatU3(1-198aa)-GFP showed a homogeneous distribution in the cytoplasm of all cells. The N-terminal AVIKRRL-containing sequence may function in intact PatU3, as a separate peptide, or both. IMPORTANCE PatU (or split into PatU5 and PatU3) is distributed in almost all filamentous cyanobacteria, including those that do not form heterocysts (except Pseudanabaena); however, its functions other than heterocyst differentiation/patterning have not been reported before. In this study, we found that PatU3 in Anabaena sp. PCC 7120 is involved in cell size determination. The N-terminal 16-aa sequence of PatU3 is required for control of cell size and interaction with the cell division protein Ftn6, and an octapeptide (aa7 ∼ aa14) within the 16-aa sequence can restore the cell size (and heterocyst frequency) of a patU3 deletion mutant to normal. Such a peptide, if generated from PatU or PatU3 in vivo, may promote intercellular coordination in filamentous cyanobacteria.

patD, a Gene Regulated by NtcA, Is Involved in the Optimization of Heterocyst Frequency in the Cyanobacterium Anabaena sp. Strain PCC 7120

ABSTRACT In the filamentous multicellular cyanobacterium Anabaena sp. strain PCC 7120, 5 to 10% of the cells differentiate into heterocysts, which are specialized in N2 fixation. Heterocysts and vegetative cells are mutually dependent for filament growth through nutrient exchange. Thus, the heterocyst frequency should be optimized to maintain the cellular carbon and nitrogen (C/N) balance for filament fitness in the environment. Here, we report the identification of patD, whose expression is directly activated in developing cells by the transcription factor NtcA. The inactivation of patD increases heterocyst frequency and promotes the upregulation of the positive regulator of heterocyst development hetR, whereas its overexpression decreases the heterocyst frequency. The change in heterocyst frequency resulting from the inactivation of patD leads to the reduction in competitiveness of the filaments under combined-nitrogen-depleted conditions. These results indicate that patD regulates heterocyst frequency in Anabaena sp. PCC 7120, ensuring its optimal filament growth. IMPORTANCE Microorganisms have evolved various strategies in order to adapt to the environment and compete with other organisms. Heterocyst differentiation is a prokaryotic model for studying complex cellular regulation. The NtcA-regulated gene patD controls the ratio of heterocysts relative to vegetative cells on the filaments of Anabaena sp. strain PCC 7120. Such a regulation provides a mechanism through which carbon fixation by vegetative cells and nitrogen fixation by heterocysts are properly balanced to ensure optimal growth and keep a competitive edge for long-term survival.

Do the regular annual extreme water level changes affect the seasonal appearance of Anabaena in Poyang Lake?

Background Poyang Lake is an ecosystem experiencing annual variations in water level of up to 14 m. Water level changes were 8.03 and 11.22 m, respectively, in the years 2013 and 2014. The biomass and heterocyst frequency of Anabaena increased in the summers of recent years. Methods A weekly to bi-weekly monitoring from June to November 2013 and 2014 was set up to explain the variations of Anabaena appearance in different phases of the water level. Results Anabaena was present in the lake throughout the year. The average relative biomass of Anabaena in the present study was over 40%, being most abundant in summer. The average heterocyst frequency was 0.23% in 2013 and 0.76% in 2014. Correlation analysis indicated a positive trend of Anabaena biomass with water temperature and water level and a negative one with total nitrogen (TN), which is the reason for the increase of heterocyst frequency in 2013 and 2014. Heterocyst frequency of Anabaena was positively correlated with water temperature, water level and PO4-P, and negatively with dissolved inorganic nitrogen (DIN/DIP), NO3-N and TN. Moreover, water temperature and DIN/DIP were significantly correlated with water level, indicating that water level changes have a direct effect on Anabaena and heterocyst formation in Poyang Lake. Conclusions The results of this study support the hypothesis that increasing biomass and heterocyst formation of Anabaena can be primarily caused by seasonal changes of the water level in Poyang Lake.

Do the regular annual extreme water level changes affect the seasonal appearance of Anabaena in Poyang Lake?

Background. Poyang Lake is an exceptional system exhibiting a water level change of up to 14 m every year. In the years 2013 and 2014 water level changes were 8.03 m and 11.22m respectively. The biomass of Anabaena and heterocyst frequency increased in the summers of recent years. Methods. A weekly or bi-weekly monitoring period from June to November 2013 and 2014 was set up to explain the reason for this phenomenon. Results. Anabaena was observed throughout the year. The average relative biomass of Anabaena in the study period was over 40%, being most abundant in summer. The average heterocyst frequency was 0.226% in 2013 and 0.760% in 2014. The SPSS analysis indicated a positive correlation of Anabaena biomass with water temperature and water level and negative one with TN, which is the reason for the increase of heterocyst frequency from 2013 to 2014. It also indicated that heterocyst frequency of Anabaena was positively correlated with water temperature, water level and PO4-P, and negatively with DIN/DIP, NO3-N and TN. At the same time, water temperature and DIN/DIP were significantly correlated with water level, which means that water level changes have a direct effect on Anabaena and its heterocyst formation in Poyang Lake. Conclusions. The results of the study support the hypothesis that increasing biomass and heterocyst formation of Anabaena were primarily caused by changes in the environmental parameters, such as high water temperature and suitable nutrients in summer and autumn, driven by the regular annual extreme water level change of Poyang Lake. The results can provide a theoretical basis for the management and utilization of Lake Poyang.

Do the regular annual extreme water level changes affect the seasonal appearance of Anabaena in Poyang Lake?

Background. Poyang Lake is an exceptional system exhibiting a water level change of up to 14 m every year. In the years 2013 and 2014 water level changes were 8.03 m and 11.22m respectively. The biomass of Anabaena and heterocyst frequency increased in the summers of recent years. Methods. A weekly or bi-weekly monitoring period from June to November 2013 and 2014 was set up to explain the reason for this phenomenon. Results. Anabaena was observed throughout the year. The average relative biomass of Anabaena in the study period was over 40%, being most abundant in summer. The average heterocyst frequency was 0.226% in 2013 and 0.760% in 2014. The SPSS analysis indicated a positive correlation of Anabaena biomass with water temperature and water level and negative one with TN, which is the reason for the increase of heterocyst frequency from 2013 to 2014. It also indicated that heterocyst frequency of Anabaena was positively correlated with water temperature, water level and PO4-P, and negatively with DIN/DIP, NO3-N and TN. At the same time, water temperature and DIN/DIP were significantly correlated with water level, which means that water level changes have a direct effect on Anabaena and its heterocyst formation in Poyang Lake. Conclusions. The results of the study support the hypothesis that increasing biomass and heterocyst formation of Anabaena were primarily caused by changes in the environmental parameters, such as high water temperature and suitable nutrients in summer and autumn, driven by the regular annual extreme water level change of Poyang Lake. The results can provide a theoretical basis for the management and utilization of Lake Poyang.

The Anabaena sp. Strain PCC 7120 Gene all2874 Encodes a Diguanylate Cyclase and Is Required for Normal Heterocyst Development under High-Light Growth Conditions

ABSTRACT The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 harbors 14 genes containing a GGDEF diguanylate cyclase domain. We found that inactivation of one of these genes, all2874, caused abnormal heterocyst development. The all2874 mutant showed a pronounced reduction in heterocyst frequency during diazotrophic growth and reduced vegetative cell size compared to the wild type. The severity of the mutant phenotype varied with light intensity; at high light intensity, the mutant phenotype was accentuated, whereas at low light intensity the phenotype was similar to wild type. Under high-light growth conditions, the initial heterocyst frequency and pattern for the all2874 mutant were normal, but within 4 days following nitrogen step-down, many intervals between heterocysts increased to as many as 200 vegetative cells, whereas in the wild type the intervals were less than 25 vegetative cells. Filaments containing these unusually long vegetative cell intervals between heterocysts also contained intervals of normal length. An all2874 mutant strain carrying a P patS -gfp transcriptional reporter fusion failed to show normal upregulation of the reporter, which indicates that the decrease in heterocyst frequency is due to an early block in differentiation before induction of the patS gene, which in the wild type takes place 8 h after nitrogen step-down. Genetic epistasis experiments suggest that All2874 acts upstream of the master regulator HetR in differentiating cells. We also showed that purified All2874 functions as a diguanylate cyclase in vitro. We hypothesize that All2874 is required for the normal regulation of heterocyst frequency under high-light growth conditions.