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.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

In vivo Inhibition of the 3-Dehydroquinate Synthase by 7-Deoxysedoheptulose Depends on Promiscuous Uptake by Sugar Transporters in Cyanobacteria.

7-Deoxysedoheptulose (7dSh) is a bioactive deoxy-sugar actively excreted by the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) but also Streptomyces setonensis. In our previous publications we have shown that in S. elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved. Additionally, we showed that 7dSh inhibits the growth of cyanobacteria, but also the growth of plants and fungi, presumably by inhibiting the 3-dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, as the substrate of this enzyme strongly accumulates in cells treated with 7dSh. In this study, by using purified DHQS of Anabaena variabilis ATCC 29413 (A. variabilis) we biochemically confirmed that 7dSh is a competitive inhibitor of this enzyme. By analyzing the effect of 7dSh on a subset of cyanobacteria from all the five subsections, we identified different species whose growth was inhibited by 7dSh. We also found that in some of the susceptible cyanobacteria import of 7dSh is mediated by structurally different and promiscuous transporters: 7dSh can be taken up by the fructose ABC-transporter in A. variabilis and via the glucose permease in Synechocystis sp. PCC 6803 (Synechocystis sp.). In both cases, an effective uptake and thereby intracellular enrichment of 7dSh was essential for the inhibitory activity. Importantly, spontaneous mutations in the sugar transporters of A. variabilis and Synechocystis sp. not only disabled growth of the two strains on fructose and glucose, respectively, but also almost abolished their sensitivity to 7dSh. Although we have clearly shown in these examples that the effective uptake plays an essential role in the inhibitory effect of 7dSh, questions remain about how 7dSh resistance works in other (cyano)bacteria. Also, the involvement of a putative ribokinase in 7dSh resistance in the producer strain S. elongatus remained to be further investigated. Overall, these data establish 7dSh as the first allelochemical targeting the shikimate pathway in other cyanobacteria and plants and suggest a role of 7dSh in niche competition.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.