Convergent reductive evolution of cyanobacteria in symbiosis with Dinophysiales dinoflagellates.

The diversity of marine cyanobacteria has been extensively studied due to their vital roles in ocean primary production. However, little is understood about the diversity of cyanobacterial species involved in symbiotic relationships. In this study, we successfully sequenced the complete genome of a cyanobacterium in symbiosis with Citharistes regius, a dinoflagellate species thriving in the open ocean. A phylogenomic analysis revealed that the cyanobacterium (CregCyn) belongs to the marine picocyanobacterial lineage, akin to another cyanobacterial symbiont (OmCyn) of a different dinoflagellate closely related to Citharistes. Nevertheless, these two symbionts are representing distinct lineages, suggesting independent origins of their symbiotic lifestyles. Despite the distinct origins, the genome analyses of CregCyn revealed shared characteristics with OmCyn, including an obligate symbiotic relationship with the host dinoflagellates and a degree of genome reduction. In contrast, a detailed analysis of genome subregions unveiled that the CregCyn genome carries genomic islands that are not found in the OmCyn genome. The presence of the genomic islands implies that exogenous genes have been integrated into the CregCyn genome at some point in its evolution. This study contributes to our understanding of the complex history of the symbiosis between dinoflagellates and cyanobacteria, as well as the genomic diversity of marine picocyanobacteria.

Monitoring of toxic cyanobacterial blooms in Lalla Takerkoust reservoir by satellite imagery and microcystin transfer to surrounding farms.

Cyanobacterial harmful algal blooms (CyanoHABs) threaten public health and freshwater ecosystems worldwide. In this study, our main goal was to explore the dynamics of cyanobacterial blooms and how microcystins (MCs) move from the Lalla Takerkoust reservoir to the nearby farms. We used Landsat imagery, molecular analysis, collecting and analyzing physicochemical data, and assessing toxins using HPLC. Our investigation identified two cyanobacterial species responsible for the blooms: Microcystis sp. and Synechococcus sp. Our Microcystis strain produced three MC variants (MC-RR, MC-YR, and MC-LR), with MC-RR exhibiting the highest concentrations in dissolved and intracellular toxins. In contrast, our Synechococcus strain did not produce any detectable toxins. To validate our Normalized Difference Vegetation Index (NDVI) results, we utilized limnological data, including algal cell counts, and quantified MCs in freeze-dried Microcystis bloom samples collected from the reservoir. Our study revealed patterns and trends in cyanobacterial proliferation in the reservoir over 30 years and presented a historical map of the area of cyanobacterial infestation using the NDVI method. The study found that MC-LR accumulates near the water surface due to the buoyancy of Microcystis. The maximum concentration of MC-LR in the reservoir water was 160 µg L-1. In contrast, 4 km downstream of the reservoir, the concentration decreased by a factor of 5.39 to 29.63 µgL-1, indicating a decrease in MC-LR concentration with increasing distance from the bloom source. Similarly, the MC-YR concentration decreased by a factor of 2.98 for the same distance. Interestingly, the MC distribution varied with depth, with MC-LR dominating at the water surface and MC-YR at the reservoir outlet at a water depth of 10 m. Our findings highlight the impact of nutrient concentrations, environmental factors, and transfer processes on bloom dynamics and MC distribution. We emphasize the need for effective management strategies to minimize toxin transfer and ensure public health and safety.

Microcystin removal by microbial communities from a coastal lagoon: Influence of abiotic factors, bacterioplankton composition and estimated functions.

Toxic cyanobacterial blooms present a substantial risk to public health due to the production of secondary metabolites, notably microcystins (MCs). Microcystin-LR (MC-LR) is the most prevalent and toxic variant in freshwater. MCs resist conventional water treatment methods, persistently impacting water quality. This study focused on an oligohaline shallow lagoon historically affected by MC-producing cyanobacteria, aiming to identify bacteria capable of degrading MC and investigating the influence of environmental factors on this process. While isolated strains did not exhibit MC degradation, microbial assemblages directly sourced from lagoon water removed MC-LR within seven days at 25 ºC and pH 8.0. The associated bacterial community demonstrated an increased abundance of bacterial taxa assigned to Methylophilales, and also Rhodospirillales and Rhodocyclales to a lesser extent. However, elevated atmospheric temperatures (45 ºC) and acidification (pH 5.0 and 3.0) hindered MC-LR removal, indicating that extreme environmental changes could contribute to prolonged MC persistence in the water column. This study highlights the importance of considering environmental conditions in order to develop strategies to mitigate cyanotoxin contamination in aquatic ecosystems.

Comparative toxicology of algal cell extracts and pure cyanotoxins: insights into toxic effects and mechanisms of harmful cyanobacteria Raphidiopsis raciborskii.

Ongoing research on cyanotoxins, driven by the socioeconomic impact of harmful algal blooms, emphasizes the critical necessity of elucidating the toxicological profiles of algal cell extracts and pure toxins. This study comprehensively compares Raphidiopsis raciborskii dissolved extract (RDE) and cylindrospermopsin (CYN) based on Daphnia magna assays. Both RDE and CYN target vital organs and disrupt reproduction, development, and digestion, thereby causing acute and chronic toxicity. Disturbances in locomotion, reduced behavioral activity, and weakened swimming capability in D. magna have also been reported for both RDE and CYN, indicating the insufficiency of conventional toxicity evaluation parameters for distinguishing between the toxic effects of algal extracts and pure cyanotoxins. Additionally, chemical profiling revealed the presence of highly active tryptophan-, humic acid-, and fulvic acid-like fluorescence compounds in the RDE, along with the active constituents of CYN, within a 15-day period, demonstrating the chemical complexity and dynamics of the RDE. Transcriptomics was used to further elucidate the distinct molecular mechanisms of RDE and CYN. They act diversely in terms of cytotoxicity, involving oxidative stress and response, protein content, and energy metabolism, and demonstrate distinct modes of action in neurofunctions. In essence, this study underscores the distinct toxicity mechanisms of RDE and CYN and emphasizes the necessity for context- and objective-specific toxicity assessments, advocating nuanced approaches to evaluate the ecological and health implications of cyanotoxins, thereby contributing to the precision of environmental risk assessments.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Recognition of cyanobacteria promoters via Siamese network-based contrastive learning under novel non-promoter generation.

It is a vital step to recognize cyanobacteria promoters on a genome-wide scale. Computational methods are promising to assist in difficult biological identification. When building recognition models, these methods rely on non-promoter generation to cope with the lack of real non-promoters. Nevertheless, the factitious significant difference between promoters and non-promoters causes over-optimistic prediction. Moreover, designed for E. coli or B. subtilis, existing methods cannot uncover novel, distinct motifs among cyanobacterial promoters. To address these issues, this work first proposes a novel non-promoter generation strategy called phantom sampling, which can eliminate the factitious difference between promoters and generated non-promoters. Furthermore, it elaborates a novel promoter prediction model based on the Siamese network (SiamProm), which can amplify the hidden difference between promoters and non-promoters through a joint characterization of global associations, upstream and downstream contexts, and neighboring associations w.r.t. k-mer tokens. The comparison with state-of-the-art methods demonstrates the superiority of our phantom sampling and SiamProm. Both comprehensive ablation studies and feature space illustrations also validate the effectiveness of the Siamese network and its components. More importantly, SiamProm, upon our phantom sampling, finds a novel cyanobacterial promoter motif ('GCGATCGC'), which is palindrome-patterned, content-conserved, but position-shifted.

Physico-chemical characterisation of irrigation basin waters and inventory study of their algal communities.

To cope with the water shortage in Sous Massa region of Morocco, agricultural producers in the region have resorted to different types of water supply basins, known as "irrigation basins" but the phenomenon of eutrophication has hindered the continuity of agricultural productivity by altering the quality of the water used for irrigation on the one hand, and causing economic damage to agricultural producers due to the clogging of the water pumping network on the other. We began by characterising the physico-chemical quality of the water to determine the causes of its high nutrient content, then we determined the taxonomy of the algal species in the irrigation basins to which we had access. A qualitative study of the water in the irrigation basins in order to better explain the inventory obtained from the taxonomic identification of the algal biomass collected, which proved the existence of new species, not previously identified, characterising the freshwaters of the Moroccan region, is under the scope of this work. The species studied belong mainly to the following groups: green algae (11 genera of Chlorophyta and 7 genera of Charophyta), blue algae (7 genera of Cyanobacteria), brown algae (7 genera of Diatoms), and one genus of Euglenophyta.

How do seasonal temperature variations influence interplay between toxic and non-toxic cyanobacterial blooms? Evidence from modeling and experimental data.

Summer cyanobacterial blooms exhibit a dynamic interplay between toxic and non-toxic genotypes, significantly influencing the cyanotoxin levels within a lake. The challenge lies in accurately predicting these toxin concentrations due to the significant temporal fluctuations in the proportions of toxic and non-toxic genotypes. Typically, the toxic genotypes dominate during the early and late summer periods, while the non-toxic variants prevail in mid-summer. To dissect this phenomenon, we propose a model that accounts for the competitive interaction between toxic and non-toxic genotypes, as well as seasonal temperature variations. Our numerical simulations suggest that the optimal temperature of the toxic genotypes is lower than that of the optimal temperatures of the non-toxic counterparts. This difference of optimal temperature may potentially contribute to explain the dominance of toxic genotypes at the early and late summer periods, situation often observed in the field. Experimental data from the laboratory align qualitatively with our simulation results, enabling a better understanding of complex interplays between toxic and non-toxic cyanobacteria.

Spatio-temporal connectivity of a toxic cyanobacterial community and its associated microbiome along a freshwater-marine continuum.

Due to climate changes and eutrophication, blooms of predominantly toxic freshwater cyanobacteria are intensifying and are likely to colonize estuaries, thus impacting benthic organisms and shellfish farming representing a major ecological, health and economic risk. In the natural environment, Microcystis form large mucilaginous colonies that influence the development of both cyanobacterial and embedded bacterial communities. However, little is known about the fate of natural colonies of Microcystis by salinity increase. In this study, we monitored the fate of a Microcystis dominated bloom and its microbiome along a French freshwater-marine gradient at different phases of a bloom. We demonstrated changes in the cyanobacterial genotypic composition, in the production of specific metabolites (toxins and compatible solutes) and in the heterotrophic bacteria structure in response to the salinity increase. In particular M. aeruginosa and M. wesenbergii survived salinities up to 20. Based on microcystin gene abundance, the cyanobacteria became more toxic during their estuarine transfer but with no selection of specific microcystin variants. An increase in compatible solutes occurred along the continuum with extensive trehalose and betaine accumulations. Salinity structured most the heterotrophic bacteria community, with an increased in the richness and diversity along the continuum. A core microbiome in the mucilage-associated attached fraction was highly abundant suggesting a strong interaction between Microcystis and its microbiome and a likely protecting role of the mucilage against an osmotic shock. These results underline the need to better determine the interactions between the Microcystis colonies and their microbiome as a likely key to their widespread success and adaptation to various environmental conditions.

Lactic acid fermented microalgae and cyanobacteria as a new source of lipid reducing compounds: assessment through zebrafish Nile red fat metabolism assay and untargeted metabolomics.

Obesity is one of the most important threats to human health. Besides existing pharmacological or clinical interventions, novel effective and largely available solutions are still necessary. Among diverse natural resources, microalgae are well known for their complexity in the production of novel secondary metabolites. At the same time, lactic acid bacteria (LAB) are known for their capacity to metabolize, through fermentation, different matrices, and consequently to modify or produce new compounds with potential bioactivity. This work aimed to study the production of fermented microalgae and cyanobacteria, and to analyse their extracts in the zebrafish Nile red fat metabolism assay. Three microalgal species (Chlorella vulgaris, Chlorococcum sp. and Arthrospira platensis) were fermented with seven strains of LAB from 4 species (Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii bulgaricus and Lacticaseibacillus paracasei), derived from the UPCCO - University of Parma Culture Collection, Parma, Italy). All the selected strains were able to ferment the selected species of microalgae, and the most suitable substrate for LAB growth was Arthrospira platensis. Extracts from fermented Chlorella vulgaris and Chlorococcum sp. reduced significantly the neutral lipid reservoirs, which was not observed without fermentations. The strongest lipid reducing effect was obtained with Arthrospira platensis fermented with Lactobacillus delbrueckii bulgaricus 1932. Untargeted metabolomics identified some compound families, which could be related to the observed bioactivity, namely fatty acids, fatty amides, triterpene saponins, chlorophyll derivatives and purine nucleotides. This work opens up the possibility of developing novel functional foods or food supplements based on microalgae, since lactic acid fermentation enhanced the production of bioactive compounds with lipid reducing activities.

Comprehensive analysis of cyanobacterial secondary metabolites distribution and toxicity in urban water bodies.

This study addresses the increasing concern regarding cyanotoxin contamination of water bodies, highlighting the diversity of these toxins and their potential health implications. Cyanobacteria, which are prevalent in aquatic environments, produce toxic metabolites, raising concerns regarding human exposure and associated health risks, including a potential increase in cancer risk. Although existing research has primarily focused on well-known cyanotoxins, recent technological advancements have revealed numerous unknown cyanotoxins, necessitating a comprehensive assessment of multiple toxin categories. To enhance the cyanotoxin databases, we optimized the CyanoMetDB cyanobacterial secondary metabolites database by incorporating secondary fragmentation patterns using the Mass Frontier fragmentation data prediction software. Water samples from diverse locations in Shanghai were analyzed using high-resolution mass spectrometry. Subsequently, the toxicity of cyanobacterial metabolites in the water samples was examined through acute toxicity assays using the crustacean Thamnocephalus platyurus. After 24 h of exposure, the semi-lethal concentrations (LC50) of the water samples ranged from 0.31 mg L-1 to 1.78 mg L-1 (MC-LR equivalent concentration). Our findings revealed a critical correlation between the overall concentration of cyanobacterial metabolites and toxicity. The robust framework and insights of this study underscore the need for an inclusive approach to water quality management, emphasizing continuous efforts to refine detection methods and comprehend the broader ecological impact of cyanobacterial blooms on aquatic ecosystems.

A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

Direct interaction between marine cyanobacteria mediated by nanotubes.

Microbial associations and interactions drive and regulate nutrient fluxes in the ocean. However, physical contact between cells of marine cyanobacteria has not been studied thus far. Here, we show a mechanism of direct interaction between the marine cyanobacteria Prochlorococcus and Synechococcus, the intercellular membrane nanotubes. We present evidence of inter- and intra-genus exchange of cytoplasmic material between neighboring and distant cells of cyanobacteria mediated by nanotubes. We visualized and measured these structures in xenic and axenic cultures and in natural samples. We show that nanotubes are produced between living cells, suggesting that this is a relevant system of exchange material in vivo. The discovery of nanotubes acting as exchange bridges in the most abundant photosynthetic organisms in the ocean may have important implications for their interactions with other organisms and their population dynamics.

The modular architecture of sigma factors in cyanobacteria: a framework to assess their diversity and understand their evolution.

BACKGROUND: Bacterial RNA polymerase holoenzyme requires sigma70 factors to start transcription by identifying promoter elements. Cyanobacteria possess multiple sigma70 factors to adapt to a wide variety of ecological niches. These factors are grouped into two categories: primary sigma factor initiates transcription of housekeeping genes during normal growth conditions, while alternative sigma factors initiate transcription of specific genes under particular conditions. However, the present classification does not consider the modular organization of their structural domains, introducing therefore multiple functional and structural biases. A comprehensive analysis of this protein family in cyanobacteria is needed to address these limitations. RESULTS: We investigated the structure and evolution of sigma70 factors in cyanobacteria, analyzing their modular architecture and variation among unicellular, filamentous, and heterocyst-forming morphotypes. 4,193 sigma70 homologs were found with 59 distinct modular patterns, including six essential and 29 accessory domains, such as DUF6596. 90% of cyanobacteria typically have 5 to 17 sigma70 homologs and this number likely depends on the strain morphotype, the taxonomic order and the genome size. We classified sigma70 factors into 12 clans and 36 families. According to taxonomic orders and phenotypic traits, the number of homologs within the 14 main families was variable, with the A.1 family including the primary sigma factor since this family was found in all cyanobacterial species. The A.1, A.5, C.1, E.1, J.1, and K.1 families were found to be key sigma families that distinguish heterocyst-forming strains. To explain the diversification and evolution of sigma70, we propose an evolutionary scenario rooted in the diversification of a common ancestor of the A1 family. This scenario is characterized by evolutionary events including domain losses, gains, insertions, and modifications. The high occurrence of the DUF6596 domain in bacterial sigma70 proteins, and its association with the highest prevalence observed in Actinobacteria, suggests that this domain might be important for sigma70 function. It also implies that the domain could have emerged in Actinobacteria and been transferred through horizontal gene transfer. CONCLUSION: Our analysis provides detailed insights into the modular domain architecture of sigma70, introducing a novel robust classification. It also proposes an evolutionary scenario explaining their diversity across different taxonomical orders.

Cyanotoxin Occurrence and Diversity in 98 Cyanobacterial Blooms from Swedish Lakes and the Baltic Sea.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

Microbial Community Response to Granular Peroxide-Based Algaecide Treatment of a Cyanobacterial Harmful Algal Bloom in Lake Okeechobee, Florida (USA).

Cyanobacterial harmful algal blooms (cyanoHABs) occur in fresh water globally. These can degrade water quality and produce toxins, resulting in ecological and economic damages. Thus, short-term management methods (i.e., algaecides) are necessary to rapidly mitigate the negative impacts of cyanoHABs. In this study, we assess the efficacy of a hydrogen peroxide-based algaecide (PAK® 27) on a Microcystis dominated bloom which occurred within the Pahokee Marina on Lake Okeechobee, Florida, USA. We observed a significant reduction in chlorophyll a (96.81%), phycocyanin (93.17%), and Microcystis cell counts (99.92%), and a substantial reduction in microcystins (86.7%) 48 h after treatment (HAT). Additionally, there was a significant shift in bacterial community structure 48 HAT, which coincided with an increase in the relative abundance of photosynthetic protists. These results indicate that hydrogen peroxide-based algaecides are an effective treatment method for cyanoHAB control and highlight their effects on non-target microorganisms (i.e., bacteria and protists).

Characterization of Neowestiellopsis persica A1387 (Hapalosiphonaceae) based on the cpcA, psbA, rpoC1, nifH and nifD gene sequences.

BACKGROUND: Complex descriptions of new strains of cyanobacteria appear very frequently. The main importance of these descriptions concerns potential new substances that they could synthesise, as well as their different properties as a result of their different ecological niches. The main gene used for these descriptions is 16 S with ITS or whole genome sequencing. Neowestiellopsis persica represents a unique example of the influence of ecology on morphological changes, with almost identical 16 S identity. Although our previously described Neowestiellopsis persica strain A1387 was characterized by 16 S analysis, we used different molecular markers to provide a way to separate strains of this genus that are closely related at the genetic level. MATERIALS AND METHODS: In order to conduct an in-depth study, several molecular markers, namely psbA, rpoC1, nifD, nifH and cpcA were sequenced and studied in Neowestiellopsis persica strain A1387. RESULTS: The results of the phylogenetic analysis, based on cpcA, showed that the studied strain A 1387 falls into a separate clade than N. persica, indicating that this signature sequence could be a useful molecular marker for phylogenetic separation of similar strains isolated in the future. CONCLUSIONS: Analysis of strain A1387 based on gene differences confirmed that it is a Neowestiellopsis strain. The morphological changes observed in the previous study could be due to different ecological and cultivation conditions compared to the type species. At the same time, the sequences obtained have increased our understanding of this species and will help in the future to better identify strains belonging to the genus Neowestiellopsis.

Investigating the Balance between Structural Conservation and Functional Flexibility in Photosystem I.

Photosynthesis, as the primary source of energy for all life forms, plays a crucial role in maintaining the global balance of energy, entropy, and enthalpy in living organisms. Among its various building blocks, photosystem I (PSI) is responsible for light-driven electron transfer, crucial for generating cellular reducing power. PSI acts as a light-driven plastocyanin-ferredoxin oxidoreductase and is situated in the thylakoid membranes of cyanobacteria and the chloroplasts of eukaryotic photosynthetic organisms. Comprehending the structure and function of the photosynthetic machinery is essential for understanding its mode of action. New insights are offered into the structure and function of PSI and its associated light-harvesting proteins, with a specific focus on the remarkable structural conservation of the core complex and high plasticity of the peripheral light-harvesting complexes.

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a "false positive", the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.