Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis.

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In free-living Rlv3841, the hFixL-FxkR-FixK pathway was active at 1% O2, and confocal microscopy showed hFixL-FxkR-FixK activity in the earliest stages of Rlv3841 differentiation in nodules (zones I and II). Work on Rlv3841 inside and outside nodules showed that the hFixL-FxkR-FixK pathway also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III and IV of pea nodules. Quantification of fixNOQP expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK pathway effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In zone III of mature nodules, near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required for wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation is prevalent in rhizobia, suggesting the fine-tuned control this enables is common and maximizes the effectiveness of the symbioses.

COVID-19, SARS-CoV-2, and Export Controls.

Export controls are intended to prevent the proliferation of materials that could be misused to make biological weapons. They are not intended to stifle critical research and development in the midst of a pandemic. This article explores how and why export controls might apply to severe acute respiratory syndrome coronavirus 2, the virus that causes coronavirus disease 2019. It outlines the taxonomic and genetic factors associated with the current approach to export control lists and discusses how they lead to unnecessary ambiguity. The authors describe ways in which the current export control systems might be revised in the short, medium, and long term, including sequence, disease, and function-based approaches.

Global control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch.

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance.

Oxygen regulatory mechanisms of nitrogen fixation in rhizobia.

Rhizobia are α- and ß-proteobacteria that form a symbiotic partnership with legumes, fixing atmospheric dinitrogen to ammonia and providing it to the plant. Oxygen regulation is key in this symbiosis. Fixation is performed by an oxygen-intolerant nitrogenase enzyme but requires respiration to meet its high energy demands. To satisfy these opposing constraints the symbiotic partners cooperate intimately, employing a variety of mechanisms to regulate and respond to oxygen concentration. During symbiosis rhizobia undergo significant changes in gene expression to differentiate into nitrogen-fixing bacteroids. Legumes host these bacteroids in specialized root organs called nodules. These generate a near-anoxic environment using an oxygen diffusion barrier, oxygen-binding leghemoglobin and control of mitochondria localization. Rhizobia sense oxygen using multiple interconnected systems which enable a finely-tuned response to the wide range of oxygen concentrations they experience when transitioning from soil to nodules. The oxygen-sensing FixL-FixJ and hybrid FixL-FxkR two-component systems activate at relatively high oxygen concentration and regulate fixK transcription. FixK activates the fixNOQP and fixGHIS operons producing a high-affinity terminal oxidase required for bacterial respiration in the microaerobic nodule. Additionally or alternatively, some rhizobia regulate expression of these operons by FnrN, an FNR-like oxygen-sensing protein. The final stage of symbiotic establishment is activated by the NifA protein, regulated by oxygen at both the transcriptional and protein level. A cross-species comparison of these systems highlights differences in their roles and interconnections but reveals common regulatory patterns and themes. Future work is needed to establish the complete regulon of these systems and identify other regulatory signals.

High speed two-dimensional optical beam position detector.

Disclosed is the design of a high speed two-dimensional optical beam position detector which outputs the X and Y displacement and total intensity linearly. The experimental detector measures the displacement from DC to 123 MHz and the intensity of an optical spot in a similar way as a conventional quadrant photodiode detector. The design uses four discrete photodiodes and simple dedicated optics for the position decomposition which enables higher spatial accuracy and faster electronic processing than conventional detectors. Measurements of the frequency response and the spatial sensitivity demonstrate high suitability for atomic force microscopy, scanning probe data storage applications, and wideband wavefront sensing. The operation principle allows for position measurements up to 20 GHz and more in bandwidth.