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A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis. [ABSTRACT FROM AUTHOR]

Investigating spatial patterns of animal occupancy and reproduction in peripheral populations can provide insight into factors that form species range boundaries. Following historical extirpation, American black bears ( Ursus americanus ) recolonized the western Great Basin in Nevada from the Sierra Nevada during the late 1900s. This range expansion, however, has not continued further into the Great Basin despite the presence of additional habitat. We aimed to quantify whether reduced reproduction toward the range edge contributes to this range boundary. We analyzed black bear detections from 100 camera traps deployed across black bear distribution in western Nevada using a multistate occupancy model that quantified the probability of occupancy and reproduction (i.e., female bears with cubs occupancy) in relation to changes in habitat type and habitat amount toward the range boundary. We detected a strong effect of habitat amount and habitat type on the probability of black bear occupancy and reproduction. At similar levels of landscape-scale habitat amount (e.g., 50%), estimated probability of occupancy for adult bears in piñon-juniper woodlands near the range boundary was 0.39, compared to ~1.0 in Sierra Nevada mixed-conifer forest (i.e., core habitat). Furthermore, estimated probability of cub occupancy, conditional on adult bear occupancy, in landscapes with 50% habitat was 0.32 in Great Basin piñon-juniper woodlands, compared to 0.92 in Sierra Nevada mixed-conifer forest. Black bear range in the western Great Basin conforms to the center-periphery hypothesis, with piñon-juniper woodland at the range edge supporting ecologically marginal habitat for the species compared to habitat in the Sierra Nevada. Further geographic expansion of black bears in the Great Basin may be limited by lower occupancy of reproducing females in piñon-juniper woodland. Center-periphery range dynamics may be common in large carnivore species, as their dispersal ability allows them to colonize low-quality habitat near range edges., Competing Interests: The authors have no competing interests to disclose., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: Chikungunya, a mosquito-borne viral disease caused by the chikungunya virus (CHIKV), causes a significant global health burden, and there is currently no approved vaccine to prevent chikungunya disease. In this study, the safety and immunogenicity of a CHIKV mRNA vaccine candidate (mRNA-1388) were evaluated in healthy participants in a CHIKV-nonendemic region., Methods: This phase 1, first-in-human, randomized, placebo-controlled, dose-ranging study enrolled healthy adults (ages 18-49 years) between July 2017 and March 2019 in the United States. Participants were randomly assigned (3:1) to receive 2 intramuscular injections 28 days apart with mRNA-1388 in 3 dose-level groups (25 μg, 50 μg, and 100 μg) or placebo and were followed for up to 1 year. Safety (unsolicited adverse events [AEs]), tolerability (local and systemic reactogenicity; solicited AEs), and immunogenicity (geometric mean titers [GMTs] of CHIKV neutralizing and binding antibodies) of mRNA-1388 versus placebo were evaluated., Results: Sixty participants were randomized and received ≥ 1 vaccination; 54 (90 %) completed the study. mRNA-1388 demonstrated favorable safety and reactogenicity profiles at all dose levels. Immunization with mRNA-1388 induced substantial and persistent humoral responses. Dose-dependent increases in neutralizing antibody titers were observed; GMTs (95 % confidence intervals [CIs]) at 28 days after dose 2 were 6.2 (5.1-7.6) for mRNA-1388 25 μg, 53.8 (26.8-108.1) for mRNA-1388 50 μg, 92.8 (43.6-197.6) for mRNA-1388 100 μg, and 5.0 (not estimable) for placebo. Persistent humoral responses were observed up to 1 year after vaccination and remained higher than placebo in the 2 higher mRNA-1388 dose groups. The development of CHIKV-binding antibodies followed a similar trend as that observed with neutralizing antibodies., Conclusions: mRNA-1388, the first mRNA vaccine against CHIKV, was well tolerated and elicited substantial and long-lasting neutralizing antibody responses in healthy adult participants in a nonendemic region., Clinicaltrials: gov: NCT03325075., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.A., C.A.S., C.K., H.Z., and L.P. are employees of Moderna, Inc., and may hold stock/stock options in the company. S.B., P.-G.J.B., T.B., and S.W. have no relevant or material financial interests that relate to the research described in this article., (Copyright © 2023. Published by Elsevier Ltd.)

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b 6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO 2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO2 fixation into carbohydrates by the Calvin-Benson-Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b6f (cytb6f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH+). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO2 fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytochrome bc 1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b , cytochrome c 1 , and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome bc 1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc 1 complex from the purple phototrophic bacterium Rhodobacter   sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene-maleic acid copolymer to purify the R. sphaeroides cytochrome bc 1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc 1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c 1 subunits. We observe a quinone at the Q o quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.

Quantifying cellular components is a basic and important step for understanding how a cell works, how it responds to environmental changes, and for re-engineering cells to produce valuable metabolites and increased biomass. We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. Components of biosynthetic pathways, such as those for chlorophyll or for photosystem II assembly, range between 1000 and 10,000 copies per cell, but can be tenfold higher for CO 2 fixation enzymes. The most abundant subunits are those for photosystem I, with around 100,000 copies per cell, approximately 2 to fivefold higher than for photosystem II and ATP synthase, and 5-20 fold more than for the cytochrome b 6 f complex. Disparities between numbers of pathway enzymes, between components of electron transfer chains, and between subunits within complexes indicate possible control points for biosynthetic processes, bioenergetic reactions and for the assembly of multisubunit complexes., (© 2022. The Author(s).)

FtsH proteases are membrane-embedded proteolytic complexes important for protein quality control and regulation of various physiological processes in bacteria, mitochondria, and chloroplasts. Like most cyanobacteria, the model species Synechocystis sp. PCC 6803 contains four FtsH homologs, FtsH1-FtsH4. FtsH1-FtsH3 form two hetero-oligomeric complexes, FtsH1/3 and FtsH2/3, which play a pivotal role in acclimation to nutrient deficiency and photosystem II quality control, respectively. FtsH4 differs from the other three homologs by the formation of a homo-oligomeric complex, and together with Arabidopsis thaliana AtFtsH7/9 orthologs, it has been assigned to another phylogenetic group of unknown function. Our results exclude the possibility that Synechocystis FtsH4 structurally or functionally substitutes for the missing or non-functional FtsH2 subunit in the FtsH2/3 complex. Instead, we demonstrate that FtsH4 is involved in the biogenesis of photosystem II by dual regulation of high light-inducible proteins (Hlips). FtsH4 positively regulates expression of Hlips shortly after high light exposure but is also responsible for Hlip removal under conditions when their elevated levels are no longer needed. We provide experimental support for Hlips as proteolytic substrates of FtsH4. Fluorescent labeling of FtsH4 enabled us to assess its localization using advanced microscopic techniques. Results show that FtsH4 complexes are concentrated in well-defined membrane regions at the inner and outer periphery of the thylakoid system. Based on the identification of proteins that co-purified with the tagged FtsH4, we speculate that FtsH4 concentrates in special compartments in which the biogenesis of photosynthetic complexes takes place., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and β-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris , each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αβ-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αβ-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Cyanobacteria are ubiquitous in nature and have developed numerous strategies that allow them to live in a diverse range of environments. Certain cyanobacteria synthesize chlorophylls d and f to acclimate to niches enriched in far-red light (FRL) and incorporate paralogous photosynthetic proteins into their photosynthetic apparatus in a process called FRL-induced photoacclimation (FaRLiP). We characterized the macromolecular changes involved in FRL-driven photosynthesis and used atomic force microscopy to examine the supramolecular organization of photosystem I associated with FaRLiP in three cyanobacterial species. Mass spectrometry showed the changes in the proteome of Chroococcidiopsis thermalis PCC 7203 that accompany FaRLiP. Fluorescence lifetime imaging microscopy and electron microscopy reveal an altered cellular distribution of photosystem complexes and illustrate the cell-to-cell variability of the FaRLiP response.

The dimeric reaction centre light-harvesting 1 (RC-LH1) core complex of Rhodobacter sphaeroides converts absorbed light energy to a charge separation, and then it reduces a quinone electron and proton acceptor to a quinol. The angle between the two monomers imposes a bent configuration on the dimer complex, which exerts a major influence on the curvature of the membrane vesicles, known as chromatophores, where the light-driven photosynthetic reactions take place. To investigate the dimerisation interface between two RC-LH1 monomers, we determined the cryogenic electron microscopy structure of the dimeric complex at 2.9 Å resolution. The structure shows that each monomer consists of a central RC partly enclosed by a 14-subunit LH1 ring held in an open state by PufX and protein-Y polypeptides, thus enabling quinones to enter and leave the complex. Two monomers are brought together through N-terminal interactions between PufX polypeptides on the cytoplasmic side of the complex, augmented by two novel transmembrane polypeptides, designated protein-Z, that bind to the outer faces of the two central LH1 β polypeptides. The precise fit at the dimer interface, enabled by PufX and protein-Z, by C-terminal interactions between opposing LH1 αβ subunits, and by a series of interactions with a bound sulfoquinovosyl diacylglycerol lipid, bring together each monomer creating an S-shaped array of 28 bacteriochlorophylls. The seamless join between the two sets of LH1 bacteriochlorophylls provides a path for excitation energy absorbed by one half of the complex to migrate across the dimer interface to the other half., (© 2021 The Author(s).)

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Å resolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αβ heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion., (© 2021 The Author(s).)

Compared to controlled laboratory conditions, plant growth in the field is rarely optimal since it is frequently challenged by large fluctuations in light and temperature which lower the efficiency of photosynthesis and lead to photo-oxidative stress. Plants grown under natural conditions therefore place an increased onus on the regulatory mechanisms that protect and repair the delicate photosynthetic machinery. Yet, the exact changes in thylakoid proteome composition which allow plants to acclimate to the natural environment remain largely unexplored. Here, we use quantitative label-free proteomics to demonstrate that field-grown Arabidopsis plants incorporate aspects of both the low and high light acclimation strategies previously observed in laboratory-grown plants. Field plants showed increases in the relative abundance of ATP synthase, cytochrome b 6 f , ferredoxin-NADP + reductases (FNR1 and FNR2) and their membrane tethers TIC62 and TROL, thylakoid architecture proteins CURT1A, CURT1B, RIQ1, and RIQ2, the minor monomeric antenna complex CP29.3, rapidly-relaxing non-photochemical quenching (qE)-related proteins PSBS and VDE, the photosystem II (PSII) repair machinery and the cyclic electron transfer complexes NDH, PGRL1B, and PGR5, in addition to decreases in the amounts of LHCII trimers composed of LHCB1.1, LHCB1.2, LHCB1.4, and LHCB2 proteins and CP29.2, all features typical of a laboratory high light acclimation response. Conversely, field plants also showed increases in the abundance of light harvesting proteins LHCB1.3 and CP29.1, zeaxanthin epoxidase (ZEP) and the slowly-relaxing non-photochemical quenching (qI)-related protein LCNP, changes previously associated with a laboratory low light acclimation response. Field plants also showed distinct changes to the proteome including the appearance of stress-related proteins ELIP1 and ELIP2 and changes to proteins that are largely invariant under laboratory conditions such as state transition related proteins STN7 and TAP38. We discuss the significance of these alterations in the thylakoid proteome considering the unique set of challenges faced by plants growing under natural conditions., Competing Interests: The authors have no conflict of interest to declare., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Bacterial C-type haem-copper oxidases in the cbb 3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb 3 -oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni , the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential ( cj1483c and cj1486c ) or important ( cj1484c and cj1485c ) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis . Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI ( cj1155c ) and ccoS ( cj1154c ) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCu A C (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c-1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA ( cj1161c ), or copZ ( cj1162c ) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Garg, Taylor, Pastorelli, Flannery, Jackson, Johnson and Kelly.)

Apex predators can shape communities via cascading top-down effects, but the degree to which such effects depend on predator life history traits is largely unknown. Within carnivore guilds, complex hierarchies of dominance facilitate coexistence, whereby subordinate species avoid dominant counterparts by partitioning space, time, or both. We investigated whether a major life history trait (hibernation) in an apex carnivore (black bears Ursus americanus) mediated its top-down effects on the spatio-temporal dynamics of three sympatric mesocarnivore species (coyotes Canis latrans, bobcats Lynx rufus, and gray foxes Urocyon cinereoargenteus) across a 15,000 km 2 landscape in the western USA. We compared top-down, bottom-up, and environmental effects on these mesocarnivores using an integrated modeling approach. Black bears exerted top-down effects that varied as a function of hibernation and were stronger than bottom-up or environmental impacts. High black bear activity in summer and fall appeared to buffer the most subordinate mesocarnivore (gray foxes) from competition with dominant mesocarnivores (coyotes and bobcats), which were in turn released by black bear hibernation in winter and early spring. The mesocarnivore responses occurred in space (i.e., altered occupancy and site visitation intensity) rather than time (i.e., diel activity patterns unaffected). These results suggest that the spatio-temporal dynamics of mesocarnivores in this system were principally shaped by a spatial predator cascade of interference competition mediated by black bear hibernation. Thus, certain life history traits of apex predators might facilitate coexistence among competing species over broad time scales, with complex implications for lower trophic levels.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O 2 -dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP + reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min -1 , a Michaelis-Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 13 1 -hydroxy-MgPME and 13 1 -keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing., (© 2020 Wiley Periodicals LLC.)

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo-electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris A 2.65-Å resolution structure of the RC-LH1 14 -W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC Q B site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Photosynthetic acclimation, the ability to adjust the composition of the thylakoid membrane to optimise the efficiency of electron transfer to the prevailing light conditions, is crucial to plant fitness in the field. While much is known about photosynthetic acclimation in Arabidopsis, to date there has been no study that combines both quantitative label-free proteomics and photosynthetic analysis by gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy. Using these methods we investigated how the levels of 402 thylakoid proteins, including many regulatory proteins not previously quantified, varied upon long-term (weeks) acclimation of Arabidopsis to low (LL), moderate (ML) and high (HL) growth light intensity and correlated these with key photosynthetic parameters. We show that changes in the relative abundance of cytb 6 f, ATP synthase, FNR2, TIC62 and PGR6 positively correlate with changes in estimated PSII electron transfer rate and CO 2 assimilation. Improved photosynthetic capacity in HL grown plants is paralleled by increased cyclic electron transport, which positively correlated with NDH, PGRL1, FNR1, FNR2 and TIC62, although not PGR5 abundance. The photoprotective acclimation strategy was also contrasting, with LL plants favouring slowly reversible non-photochemical quenching (qI), which positively correlated with LCNP, while HL plants favoured rapidly reversible quenching (qE), which positively correlated with PSBS. The long-term adjustment of thylakoid membrane grana diameter positively correlated with LHCII levels, while grana stacking negatively correlated with CURT1 and RIQ protein abundance. The data provide insights into how Arabidopsis tunes photosynthetic electron transfer and its regulation during developmental acclimation to light intensity., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Chlorophyll synthase (ChlG) catalyses a terminal reaction in the chlorophyll biosynthesis pathway, attachment of phytol or geranylgeraniol to the C17 propionate of chlorophyllide. Cyanobacterial ChlG forms a stable complex with high light-inducible protein D (HliD), a small single-helix protein homologous to the third transmembrane helix of plant light-harvesting complexes (LHCs). The ChlG-HliD assembly binds chlorophyll, β-carotene, zeaxanthin and myxoxanthophyll and associates with the YidC insertase, most likely to facilitate incorporation of chlorophyll into translated photosystem apoproteins. HliD independently coordinates chlorophyll and β-carotene but the role of the xanthophylls, which appear to be exclusive to the core ChlG-HliD assembly, is unclear. Here we generated mutants of Synechocystis sp. PCC 6803 lacking specific combinations of carotenoids or HliD in a background with FLAG- or His-tagged ChlG. Immunoprecipitation experiments and analysis of isolated membranes demonstrate that the absence of zeaxanthin and myxoxanthophyll significantly weakens the interaction between HliD and ChlG. ChlG alone does not bind carotenoids and accumulation of the chlorophyllide substrate in the absence of xanthophylls indicates that activity/stability of the 'naked' enzyme is perturbed. In contrast, the interaction of HliD with a second partner, the photosystem II assembly factor Ycf39, is preserved in the absence of xanthophylls. We propose that xanthophylls are required for the stable association of ChlG and HliD, acting as a 'molecular glue' at the lateral transmembrane interface between these proteins; roles for zeaxanthin and myxoxanthophyll in ChlG-HliD complexation are discussed, as well as the possible presence of similar complexes between LHC-like proteins and chlorophyll biosynthesis enzymes in plants., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Distinguishing between central and peripheral causes of vertigo can be challenging not only in an acute setting but also in chronic settings. A thorough review of systems and physical exam can assist providers in differentiating central versus peripheral etiologies and the need for urgent imaging. In this case, a 47-year-old man presented with vertigo, right-sided hearing loss, right-sided headache, and right-sided facial pain that began 4 weeks before while the patient was on a cruise ship. His physical exam findings were notable for anisocoria with right pupil 3.5 and left pupil 4.5, mild ptosis on the right side, positive Romberg test to the right, and Fakuda test with deviation to the right. Urgent magnetic resonance imaging revealed dissection of the right cervical internal carotid artery with a nonocclusive intramural hematoma. This case illustrates the importance of imaging in vertigo patients in which a central etiology is suspected. It further demonstrates that isolated partial Horner's or unilateral headache may indeed be the only presenting sign in a carotid dissection., (© Association of Military Surgeons of the United States 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980's, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 μmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 μmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur., Competing Interests: The authors have declared that no competing interests exist.

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli , exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans. IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve., (Copyright © 2019 Song et al.)

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis. plantcell;31/12/2912/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light, low-light and nutrient-poor ecological niches. We used atomic force microscopy to image the different photosystem I (PSI) membrane architectures of the MED4 (high-light) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (low-light) and SS120 (low-light) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b 6 f complexes and the various Pcb proteins in the thylakoid membrane. Atomic force microscopy topographs and structural modelling revealed a series of specialized PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the low-light MIT9313 is organized into a tightly packed pseudo-hexagonal lattice that maximizes harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b 6 f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economize on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP 2- -dependent insertion of a Mg 2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA + (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg 2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA + motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA + ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH., (© 2019 The Author(s).)

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants., (© 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc 1 complex (cytbc 1 ) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc 1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc 1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc 1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc 1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b 6 f complex (cytb 6 f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc 1 /cytb 6 f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q 0 and Q i sites., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc 1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe 2+ or Mg 2+ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light-harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light-harvesting polypeptides., (© 2017 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

The North Pacific Ocean (between approximately 0°N and 50°N) contains the largest continuous ecosystem on Earth. This region plays a vital role in the cycling of globally important nutrients as well as carbon. Although the microbial communities in this region have been assessed, the dynamics of viruses (abundances and production rates) remains understudied. To address this gap, scientific cruises during the winter and summer seasons (2013) covered the North Pacific basin to determine factors that may drive virus abundances and production rates. Along with information on virus particle abundance and production, we collected a spectrum of oceanographic metrics as well as information on microbial diversity. The data suggest that both biotic and abiotic factors affect the distribution of virus particles. Factors influencing virus dynamics did not vary greatly between seasons, although the abundance of viruses was almost an order of magnitude greater in the summer. When considered in the context of microbial community structure, our observations suggest that members of the bacterial phyla Proteobacteria, Planctomycetes, and Bacteroidetes were correlated to both virus abundances and virus production rates: these phyla have been shown to be enriched in particle associated communities. The findings suggest that environmental factors influence virus community functions (e.g., virion particle degradation) and that particle-associated communities may be important drivers of virus activity.

The discovery of infectious particles that challenge conventional thoughts concerning "what is a virus" has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning "giant viruses", with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host-virus systems that fall into this "giant" category, demonstrating that this field of inquiry presents great opportunities for future research.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents that form a covalent aminal bond between their C11-position and the C2-NH 2 groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG-136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor-targeting antibodies to create antibody-drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre-clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD-containing ADCs, and explores both structure-activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)