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.

Heavy Metal Stress Alters the Response of the Unicellular Cyanobacterium Synechococcus elongatus PCC 7942 to Nitrogen Starvation

Non-diazotrophic cyanobacteria are unable to fix atmospheric nitrogen and rely on combined nitrogen for growth and development. In the absence of combined nitrogen sources, most non-diazotrophic cyanobacteria, e.g., Synechocystis sp. PCC 6803 or Synechococcus elongatus PCC 7942, enter a dormant stage called chlorosis. The chlorosis process involves switching off photosynthetic activities and downregulating protein biosynthesis. Addition of a combined nitrogen source induces the regeneration of chlorotic cells in a process called resuscitation. As heavy metals are ubiquitous in the cyanobacterial biosphere, their influence on the vegetative growth of cyanobacterial cells has been extensively studied. However, the effect of heavy metal stress on chlorotic cyanobacterial cells remains elusive. To simulate the natural conditions, we investigated the effects of long-term exposure of S. elongatus PCC 7942 cells to both heavy metal stress and nitrogen starvation. We were able to show that elevated heavy metal concentrations, especially for Ni2+, Cd2+, Cu2+ and Zn2+, are highly toxic to nitrogen starved cells. In particular, cells exposed to elevated concentrations of Cd2+ or Ni2+ were not able to properly enter chlorosis as they failed to degrade phycobiliproteins and chlorophyll a and remained greenish. In resuscitation assays, these cells were unable to recover from the simultaneous nitrogen starvation and Cd2+ or Ni2+ stress. The elevated toxicity of Cd2+ or Ni2+ presumably occurs due to their interference with the onset of chlorosis in nitrogen-starved cells, eventually leading to cell death.

Influence of environmental factors and exogenous nitrogen on nitrogen fixation by intact thallus and excised cephalodia of Peltigera aphthosa

The present study investigates the effects of various abiotic environmental factors: air humidity, moisture content, oxygen and exogenously supplied nitrogen on acetylene reduction by intact thallus and excised cephalodia of Peltgera aphthosa. Intact thallus and excised cephalodia of Peltigera aphthosa were incubated at various conditions of air humidity, moisture contents, oxygen tensions, and addition of exogenous nitrogen, and comparative nitrogen fixation by the intact thallus and excised cephalodia was used as a method for assessment. Acetylene reduction (nitrogen fixation) was enhanced at conditions of lowered oxygen tension and at initial addition of nitrogen to the medium. However, prolonged incubation of Peltigera aphthosa in the medium with combined nitrogen addition resulted in fluctuation of nitrogenase synthesis. Acetylene reduction rates were stimulated in an atmosphere of 100% relative humidity (RH) and moisture content range of 570-620% of dry weight of Peltigera aphthosa. The decrease of nitrogenase activity measurable by acetylene reduction of thallus after prolonged incubation in the medium with combined nitrogen addition shows susceptibility of the symbiosis (lichen) since the thallus showed signs of disintegration at this time. Furthermore, air humidity and moisture content of the thallus influenced nitrogenase synthesis of Peltigera aphthosa considerably on separation of cephalodia such approach decreased nitrogenase activity and also elicited differences in their response to the various treatments. Applicability of this technology would enhance plant sustainability and yield in agricultural farms.

The Inorganic Nutrient Regime and the mre Genes Regulate Cell and Filament Size and Morphology in the Phototrophic Multicellular Bacterium Anabaena

ABSTRACT The model cyanobacterium Anabaena sp. PCC 7120 exhibits a phototrophic metabolism relying on oxygenic photosynthesis and a complex morphology. The organismic unit is a filament of communicated cells that may include cells specialized in different nutritional tasks, thus representing a paradigm of multicellular bacteria. In Anabaena, the inorganic carbon and nitrogen regime influenced not only growth, but also cell size, cell shape, and filament length, which also varied through the growth cycle. When using combined nitrogen, especially with abundant carbon, cells enlarged and elongated during active growth. When fixing N2, which imposed lower growth rates, shorter and smaller cells were maintained. In Anabaena, gene homologs to mreB, mreC, and mreD form an operon that was expressed at higher levels during the phase of fastest growth. In an ntcA mutant, mre transcript levels were higher than in the wild type and, consistently, cells were longer. Negative regulation by NtcA can explain that Anabaena cells were longer in the presence of combined nitrogen than in diazotrophic cultures, in which the levels of NtcA are higher. mreB, mreC, and mreD mutants could grow with combined nitrogen, but only the latter mutant could grow diazotrophically. Cells were always larger and shorter than wild-type cells, and their orientation in the filament was inverted. Consistent with increased peptidoglycan width and incorporation in the intercellular septa, filaments were longer in the mutants, suggesting a role for MreB, MreC, and MreD in the construction of septal peptidoglycan that could affect intercellular communication required for diazotrophic growth. IMPORTANCE Most studies on the determination of bacterial cell morphology have been conducted in heterotrophic organisms. Here, we present a study of how the availability of inorganic nitrogen and carbon sources influence cell size and morphology in the context of a phototrophic metabolism, as found in the multicellular cyanobacterium Anabaena. In Anabaena, the expression of the MreB, MreC, and MreD proteins, which influence cell size and length, are regulated by NtcA, a transcription factor that globally coordinates cellular responses to the C-to-N balance of the cells. Moreover, MreB, MreC, and MreD also influence septal peptidoglycan construction, thus affecting filament length and, possibly, intercellular molecular exchange that is required for diazotrophic growth. Thus, here we identified new roles for Mre proteins in relation to the phototrophic and multicellular character of a cyanobacterium, Anabaena.

HetL provides immunity to HetR against PatS inhibition, and promotes pattern formation in the cyanobacterium Nostoc PCC 7120

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell gets immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of a heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogen (PatS), which diffusion establishes the differentiation pattern. Here we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is required to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum.