The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM.

SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.

Roles of second messengers in the regulation of cyanobacterial physiology: the carbon-concentrating mechanism and beyond.

Second messengers are a fundamental category of small molecules and ions that are involved in the regulation of many processes in all domains of life. Here we focus on cyanobacteria, prokaryotes playing important roles as primary producers in the geochemical cycles due to their capability of oxygenic photosynthesis and carbon and nitrogen fixation. Of particular interest is the inorganic carbon-concentrating mechanism (CCM), which allows cyanobacteria to concentrate CO2 near RubisCO. This mechanism needs to acclimate toward fluctuating conditions, such as inorganic carbon availability, intracellular energy levels, diurnal light cycle, light intensity, nitrogen availability, and redox state of the cell. During acclimation to such changing conditions, second messengers play a crucial role, particularly important is their interaction with the carbon control protein SbtB, a member of the PII regulator protein superfamily. SbtB is capable of binding several second messengers, uniquely adenyl nucleotides, to interact with different partners in a variety of responses. The main identified interaction partner is the bicarbonate transporter SbtA, which is regulated via SbtB depending on the energy state of the cell, the light conditions, and different CO2 availability, including cAMP signaling. The interaction with the glycogen branching enzyme, GlgB, showed a role for SbtB in the c-di-AMP-dependent regulation of glycogen synthesis during the diurnal life cycle of cyanobacteria. SbtB has also been shown to impact gene expression and metabolism during acclimation to changing CO2 conditions. This review summarizes the current knowledge about the complex second messenger regulatory network in cyanobacteria, with emphasis on carbon metabolism.

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

The impact of the cyanobacterial carbon-regulator protein SbtB and of the second messengers cAMP and c-di-AMP on CO2 -dependent gene expression.

The amount of inorganic carbon (Ci ) fluctuates in aquatic environments. Cyanobacteria evolved a Ci -concentrating mechanism (CCM) that is regulated at different levels. The regulator SbtB binds to the second messengers cAMP or c-di-AMP and is involved in acclimation to low Ci (LC) in Synechocystis sp. PCC 6803. Here, we investigated the role of SbtB and of associated second messengers at different Ci conditions. The transcriptome of wild-type (WT) Synechocystis and the ΔsbtB mutant were compared with Δcya1, a mutant defective in cAMP production, and ΔdacA, a mutant defective in generating c-di-AMP. A defined subset of LC-regulated genes in the WT was already changed in ΔsbtB under high Ci (HC) conditions. This response of ΔsbtB correlated with a diminished induction of many CCM-associated genes after LC shift in this mutant. The Δcya1 mutant showed less deviation from WT, whereas ΔdacA induced CCM-associated genes under HC. Metabolome analysis also revealed differences between the strains, whereby ΔsbtB showed slower accumulation of 2-phosphoglycolate and ΔdacA differences among amino acids compared to WT. Collectively, these results indicate that SbtB regulates a subset of LC acclimation genes while c-di-AMP and especially cAMP appear to have a lesser impact on gene expression under different Ci availabilities.

Diurnal metabolic control in cyanobacteria requires perception of second messenger signaling molecule c-di-AMP by the carbon control protein SbtB.

Because of their photosynthesis-dependent lifestyle, cyanobacteria evolved sophisticated regulatory mechanisms to adapt to oscillating day-night metabolic changes. How they coordinate the metabolic switch between autotrophic and glycogen-catabolic metabolism in light and darkness is poorly understood. Recently, c-di-AMP has been implicated in diurnal regulation, but its mode of action remains elusive. To unravel the signaling functions of c-di-AMP in cyanobacteria, we isolated c-di-AMP receptor proteins. Thereby, the carbon-sensor protein SbtB was identified as a major c-di-AMP receptor, which we confirmed biochemically and by x-ray crystallography. In search for the c-di-AMP signaling function of SbtB, we found that both SbtB and c-di-AMP cyclase­deficient mutants showed reduced diurnal growth and that c-di-AMP­bound SbtB interacts specifically with the glycogen-branching enzyme GlgB. Accordingly, both mutants displayed impaired glycogen synthesis during the day and impaired nighttime survival. Thus, the pivotal role of c-di-AMP in day-night acclimation can be attributed to SbtB-mediated regulation of glycogen metabolism.

Red/far-red light signals regulate the activity of the carbon-concentrating mechanism in cyanobacteria.

Desiccation-tolerant cyanobacteria can survive frequent hydration/dehydration cycles likely affecting inorganic carbon (Ci) levels. It was recently shown that red/far-red light serves as signal-preparing cells toward dehydration. Here, the effects of desiccation on Ci assimilation by Leptolyngbya ohadii isolated from Israel's Negev desert were investigated. Metabolomic investigations indicated a decline in ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation activity, and this was accelerated by far-red light. Far-red light negatively affected the Ci affinity of L. ohadii during desiccation and in liquid cultures. Similar effects were evident in the non-desiccation-tolerant cyanobacterium Synechocystis The Synechocystis Δcph1 mutant lacking the major phytochrome exhibited reduced photosynthetic Ci affinity when exposed to far-red light, whereas the mutant ΔsbtB lacking a Ci uptake inhibitory protein lost the far-red light inhibition. Collectively, these results suggest that red/far-red light perception likely via phytochromes regulates Ci uptake by cyanobacteria and that this mechanism contributes to desiccation tolerance in strains such as L. ohadii.

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803.

Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. Operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L-1. Likewise, yields of the sesquiterpene alcohols (-)-patchoulol and (-)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L-1 and 96.3 ± 2.2 mg * L-1, respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDC system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. Together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria.