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Abstract Background PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+. Results Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. Conclusions Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB. Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Orange carotenoid proteins (OCPs) are unique photoreceptors that are critical for cyanobacterial photoprotection. Upon exposure to blue-green light, OCPs are activated from a stable orange form, OCP O , to an active red form, OCP R , which binds to phycobilisomes (PBSs) and performs photoprotective non-photochemical quenching (NPQ). OCPs can be divided into three main families: the most abundant and best studied OCP1, and two others, OCP2 and OCP3, which have different activation and quenching properties and are yet underexplored. Crystal structures have been acquired for the three OCP clades, providing a glimpse into the conformational underpinnings of their light-absorption and energy dissipation attributes. Recently, the structure of the PBS-OCP R complex has been obtained allowing for an unprecedented insight into the photoprotective action of OCPs. Here, we review the latest findings in the field that have substantially improved our understanding of how cyanobacteria protect themselves from the toxic consequences of excess light absorption. Furthermore, current research is applying the structure of OCPs to bio-inspired optogenetic tools, to function as carotenoid delivery devices, as well as engineering the NPQ mechanism of cyanobacteria to enhance their photosynthetic biomass production., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Prochlorococcus and Synechococcus are the two most abundant photosynthetic organisms on Earth, with a strong influence on the biogeochemical carbon and nitrogen cycles. Early reports demonstrated the streamlining of regulatory mechanisms in nitrogen metabolism and the removal of genes not strictly essential. The availability of a large series of genomes, and the utilization of latest generation molecular techniques have allowed elucidating the main mechanisms developed by marine picocyanobacteria to adapt to the environments where they thrive, with a particular interest in the strains inhabiting oligotrophic oceans. Given that nitrogen is often limited in those environments, a series of studies have explored the strategies utilized by Prochlorococcus and Synechococcus to exploit the low concentrations of nitrogen-containing molecules available in large areas of the oceans. These strategies include the reduction in the GC and the cellular protein contents; the utilization of truncated proteins; a reduced average amount of N in the proteome; the development of metabolic mechanisms to perceive and utilize nanomolar nitrate concentrations; and the reduced responsiveness of key molecular regulatory systems such as NtcA to 2-oxoglutarate. These findings are in sharp contrast with the large body of knowledge obtained in freshwater cyanobacteria. We will outline the main discoveries, stressing their relevance to the ecological success of these important microorganisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

The phycobilisome is an oligomeric chromoprotein complex that serves as the principal mid-visible light-harvesting system in cyanobacteria. Here we report the observation of excitation-energy-transfer pathways involving delocalized optical excitations of the bilin (linear tetrapyrrole) chromophores in intact phycobilisomes isolated from Fremyella diplosiphon. By using broadband multidimensional electronic spectroscopy with 6.7-fs laser pulses, we are able to follow the progress of excitation energy from the phycoerythrin disks at the ends of the phycobilisome's rods to the C-phycocyanin disks along their length in <600 fs. Oscillation maps show that coherent wavepacket motions prominently involving the hydrogen out-of-plane vibrations of the bilins mediate non-adiabatic relaxation of a manifold of vibronic exciton states. However, the charge-transfer character of the bilins in the allophycocyanin-containing segments localizes the excitations in the core of the phycobilisome, yielding a kinetic bottleneck that enables photoregulatory mechanisms to operate efficiently on the >10-ps timescale., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae 1,2 . These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching 3-6 . Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties 7 , we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Marine Synechococcus , together with Prochlorococcus , contribute to a significant proportion of the primary production on Earth. The spatial distribution of these two groups of marine picocyanobacteria depends on different factors such as nutrient availability and temperature. Some Synechococcus ecotypes thrive in mesotrophic and moderately oligotrophic waters, where they exploit both oxidized and reduced forms of nitrogen. Here, we present a comprehensive study, which includes transcriptomic and proteomic analyses of the response of Synechococcus sp. strain WH7803 to nanomolar concentrations of nitrate, compared to micromolar ammonium or nitrogen starvation. We found that Synechococcus has a specific response to a nanomolar nitrate concentration that differs from the response shown under nitrogen starvation or the presence of standard concentrations of either ammonium or nitrate. This fact suggests that the particular response to the uptake of nanomolar concentrations of nitrate could be an evolutionary advantage for marine Synechococcus against Prochlorococcus in the natural environment. IMPORTANCE Marine Synechococcus are a very abundant group of photosynthetic organisms on our planet. Previous studies have shown blooms of these organisms when nanomolar concentrations of nitrate become available. We have assessed the effect of nanomolar nitrate concentrations by studying the transcriptome and proteome of Synechococcus sp. WH7803, together with some physiological parameters. We found evidence that Synechococcus sp. strain WH7803 does sense and react to nanomolar concentrations of nitrate, suggesting the occurrence of specific adaptive mechanisms to allow their utilization. Thus, very low concentrations of nitrate in the ocean seem to be a significant nitrogen source for marine picocyanobacteria.

The orange carotenoid protein (OCP) mediates nonphotochemical quenching (NPQ) mechanisms in cyanobacteria. A bound ketocarotenoid serves as a sensor of midvisible light intensity and as a quencher of phycocyanobilin excitons in the phycobilisome. The photochemical mechanism that triggers conversion of the protein from a resting, orange state (OCP O ) to an active, red state (OCP R ) after optical preparation of the S 2 state of the carotenoid remains an open question. We report here that the fluorescence spectrum and quantum yield of the bound carotenoids in OCP O report important details of the motions that follow optical preparation of the S 2 state. The fluorescence spectra from OCP O preparations containing 3'-hydroxyechinenone (3hECN) or canthaxanthin (CAN) are markedly mirror asymmetric with respect to the absorption line shape and more than an order of magnitude more intense than for carotenoids in solution. Further, 3hECN exhibits a narrower fluorescence line shape and a larger quantum yield than CAN because its excited-state motions are hindered by a hydrogen bonding interaction between the 3'-hydroxyl group on its β2 ring and Leu37 in the N-terminal domain. These results show that large-amplitude motions of the carotenoid's β2-cyclohexene ring and of the conjugated polyene backbone initiate photochemistry in OCP O .

The abundance and significant contribution to global primary production of the marine cyanobacterium Prochlorococcus have made it one of the main models in marine ecology. Several conditions known to cause strong effects on the regulation of N-related enzymes in other cyanobacteria lacked such effect in Prochlorococcus. Prochlorococcus sp. strain MIT9313 is one of the most early-branching strains among the members of this genus. In order to further understand the C/N control system in this cyanobacterium, we studied the effect of the absence of three key elements in the ocean, namely N, P and Fe, as well as the effect of inhibitors of the N assimilation or photosynthesis on the N metabolism of this strain. Furthermore, we focused our work in the effect of ageing, as the age of cultures has clear effects on the regulation of some enzymes in Prochlorococcus. To reach this goal, expression of the main three regulators involved in N assimilation in cyanobacteria, namely ntcA, glnB and pipX, as well as that of icd (encoding for isocitrate dehydrogenase) were analysed. Our results show that the control of the main proteins involved in the C/N balance in strain MIT9313 differs from other model Prochlorococcus strains., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Previous studies showed differences in the regulatory response to C/N balance in Prochlorococcus with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the ntcA gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model Prochlorococcus strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the glnA gene in the same Prochlorococcus strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in Prochlorococcus , especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching Prochlorococcus strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.

Prochlorococcus requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise knowledge of the composition and changes in the proteome can yield fundamental insights into such a response. Here we report a detailed proteome analysis of the important model cyanobacterium Prochlorococcus marinus SS120 after treatment with azaserine, an inhibitor of ferredoxin-dependent glutamate synthase (GOGAT), to simulate extreme nitrogen starvation. In total, 1,072 proteins, corresponding to 57% of the theoretical proteome, were identified-the maximum proteome coverage obtained for any Prochlorococcus strain thus far. Spectral intensity, calibrated quantification by the Hi3 method, was obtained for 1,007 proteins. Statistically significant changes ( P  value of <0.05) were observed for 408 proteins, with the majority of proteins (92.4%) downregulated after 8 h of treatment. There was a strong decrease in ribosomal proteins upon azaserine addition, while many transporters were increased. The regulatory proteins P II and PipX were decreased, and the global nitrogen regulator NtcA was upregulated. Furthermore, our data for Prochlorococcus indicate that NtcA also participates in the regulation of photosynthesis. Prochlorococcus responds to the lack of nitrogen by slowing down translation, while inducing photosynthetic cyclic electron flow and biosynthesis of proteins involved in nitrogen uptake and assimilation. IMPORTANCE Prochlorococcus is the most abundant photosynthetic organism on Earth, contributing significantly to global primary production and playing a prominent role in biogeochemical cycles. Here we study the effects of extreme nitrogen limitation, a feature of the oligotrophic oceans inhabited by this organism. Quantitative proteomics allowed an accurate quantification of the Prochlorococcus proteome, finding three main responses to nitrogen limitation: upregulation of nitrogen assimilation-related proteins, including transporters; downregulation of ribosome proteins; and induction of the photosystem II cyclic electron flow. This suggests that nitrogen limitation affects a range of metabolic processes far wider than initially believed, with the ultimate goal of saving nitrogen and maximizing the nitrogen uptake and assimilation capabilities of the cell.

The marine picocyanobacterium Synechococcus sp. WH7803 possesses two glutamine synthetases (GSs; EC 6.3.1.2), GSI encoded by glnA and GSIII encoded by glnN. This is the first work addressing the physiological regulation of both enzymes in a marine cyanobacterial strain. The increase of GS activity upon nitrogen starvation was similar to that found in other model cyanobacteria. However, an unusual response was found when cells were grown under darkness: the GS activity was unaffected, reflecting adaptation to the environment where they thrive. On the other hand, we found that GSIII did not respond to nitrogen availability, in sharp contrast with the results observed for this enzyme in other cyanobacteria thus far studied. These features suggest that GS activities in Synechococcus sp. WH7803 represent an intermediate step in the evolution of cyanobacteria, in a process of regulatory streamlining where GSI lost the regulation by light, while GSIII lost its responsiveness to nitrogen. This is in good agreement with the phylogeny of Synechococcus sp. WH7803 in the context of the marine cyanobacterial radiation.

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.