Biology subjects

Johnson, M. P.

Publications and source records attributed to Johnson, M. P..

12 recordsLinked to original sources

bioRxiv2026

Cryo-EM structures of Cereibacter sphaeroides ferrochelatase reveal an oligomeric state that occludes the substrate-binding site

Protoporphyrin IX (PPIX) is the last shared intermediate of heme and (bacterio)chlorophyll ((B)Chl) biosynthesis. Insertion of iron into PPIX by the PPIX ferrochelatase (FeCh) PpfC produces heme, whereas magnesium insertion by magnesium chelatase (MgCh) initiates (B)Chl synthesis. Chlorophototrophic organisms must regulate this biosynthetic node to ensure the correct flux of PPIX to each of these crucial tetrapyrrole products, presenting a particularly acute problem for the facultative bacterium Cereibacter (previously Rhodobacter) sphaeroides, which can switch between chemotrophic (respiratory) and phototrophic (photosynthetic) growth modes. To elucidate the structural basis for the catalytic and regulatory functions of C. sphaeroides FeCh (PpfCCs), we used cryogenic electron microscopy to determine its structure, to our knowledge the first such information for a Gram-negative or phototrophic organism. We determined the 2.6 [A] structure of an active site mutant of PpfCCs, which forms a dimer with a monomer-to-monomer interface that coordinates two hemes. We also determined a 2.5 [A] structure of the wild-type enzyme, in which two dimers interact in a homotetrameric arrangement that occludes PPIX binding at the active site of each monomer. We propose that the PpfCCs dimer likely docks onto the inner membrane in a manner analogous to the human enzyme, whereas the tetramer represents a transiently inactive state whereby the hydrophobic lips and active site are both occluded. These novel oligomerization states of FeCh may have evolved to influence the flux of tetrapyrroles through the heme/BChl branchpoint in purple bacteria.

biochemistry↗

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

HyenaSET: Hyena Sound Event Transcripts and benchmark animal2vec performance for parsing animal communication

Here, we present HyenaSET, a large (~1640 hours) bioacoustic dataset derived from collar-mounted audio recorders deployed on 19 spotted hyenas in the Maasai Mara National Reserve, Kenya. Within this dataset, 243 hours have been strongly labeled by identifying the onset and offset of all vocalizations as well as their types, and the labels have been validated by experts on hyena vocalizations and behavior. Within the strongly labeled data, the total amount of time hyenas were vocalizing was 9.5 hours (3.9%). Furthermore, each vocalization has been manually identified as "focal" (emitted by the hyena wearing the collar) or "non-focal" (emitted by a nearby conspecific), making use of information from collar-mounted accelerometers that picked up vibrations of the animals throat when it produced vocalizations. In addition to the labeled data, we also provide a large corpus of unlabeled data from the same recordings, which can be used for un- or self-supervised machine learning tasks. To ensure reproducibility of this dataset as a benchmark in machine learning studies, we present it alongside five stratified cross-validation train/test splits to enable accurate comparisons, and we also provide a train/test split in which specific individuals are left out of the training set to assess generalizability across individuals. Finally, as a performance benchmark, we present baseline results for this dataset using animal2vec, a recently developed transformer-based model optimized for bioacoustic data.

animal behavior and cognition↗

bioRxiv2025

Disequilibrium between chloroplast proton motive force and ATP levels in Arabidopsis

Current dogma holds that CO2 fixation by photosynthesis requires additional ATP production via PGR5-dependent cyclic electron transfer (PGR5-CET) to augment the NADPH and ATP produced by linear electron transfer (LET). Here we use the MgATP2- fluorescent biosensor ATeam1.03-nD/nA and find that while proton motive force (pmf) in the Arabidopsis pgr5CAS mutant is only 50-75% of that of the wild-type, MgATP2- levels are unchanged. These data demonstrate that disequilibrium exists between pmf and MgATP2- concentration in vivo, indicating that PGR5-CET is not required for ATP augmentation.

plant biology↗

bioRxiv2024

Molecular architecture of thylakoid membranes within intact spinach chloroplasts

Thylakoid membranes coordinate the light reactions of photosynthesis across multiple scales, coupling the architecture of an elaborate membrane network to the spatial organization of individual protein complexes embedded within this network. Previously, we used in situ cryo-electron tomography (cryo-ET) to reveal the native thylakoid architecture of the green alga Chlamydomonas reinhardtii [1] and then map the molecular organization of these thylakoids with single-molecule precision [2]. However, it remains to be shown how generalizable this green algal blueprint is to the thylakoids of vascular plants, which possess distinct membrane architecture subdivided into grana stacks interconnected by non-stacked stromal lamellae. Here, we continue our cryo-ET investigation to reveal the molecular architecture of thylakoids within intact chloroplasts isolated from spinach (Spinacia oleracea). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply and further develop AI-based computational approaches for automated membrane segmentation and membrane protein picking [3], enabling us to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that, despite different 3D architecture, the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. In contrast to isolated plant thylakoids, where semi-crystalline arrays of photosystem II (PSII) appear to hold some membranes together, we find in intact chloroplasts that PSII is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to C. reinhardtii, we observe strict lateral heterogeneity of PSII and PSI at the boundary between appressed and non-appressed thylakoid domains, with no evidence for a distinct grana margin region where these complexes have been proposed to intermix. Based on these measurements, we support a simple two-domain model for the molecular organization of thylakoid membranes in both green algae and plants.

plant biology↗

bioRxiv2024

Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer

Fixing CO2 via photosynthesis requires ATP and NADPH. Linear electron transfer (LET) supplies both metabolites, yet depending on environmental conditions, additional ATP is required which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is set remains largely unknown. Ferredoxin(FD)-NADP+ reductase (FNR) has been suggested to act as the switch, channelling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or CET when bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. We circumvented this problem by using CRISPR-Cas9 gene editing in Chlamydomonas reinhardtii to create a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth relative to the wild type, and decreased LET and non-photochemical quenching (NPQ) under high CO2 conditions. However, higher rates of CET and increased NPQ were found in the mutants under low CO2 or low O2 conditions where CET is normally up-regulated. Thus, rather than PSI-FNR interactions promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.

biochemistry↗

bioRxiv2023

PGR5 is required to avoid photosynthetic oscillations during light transitions

The production of ATP and NADPH by the light reactions of photosynthesis and their consumption by the Calvin-Benson-Bassham (CBB) cycle and other downstream metabolic reactions requires careful regulation. Environmental shifts perturb this careful balance, leading to photo-oxidative stress and losses in CO2 assimilation. Imbalances in the production and consumption of ATP and NADPH manifest themselves as transient instability in the chlorophyll fluorescence, P700, electrochromic shift and CO2 uptake signals recorded on leaves. These oscillations can be induced in wild-type plants by sudden shifts in CO2 concentration or light intensity, however mutants exhibiting increased oscillatory behaviour have yet to be reported. This has precluded an understanding of the regulatory mechanisms employed by plants to suppress oscillations. Here we show that the Arabidopsis pgr5 mutant, which is deficient in PGR5-dependent cyclic electron transfer (CET), exhibits increased oscillatory behaviour. In contrast, mutants lacking the NDH-dependent CET are largely unaffected. The absence of oscillations in the hope2 mutant, which like pgr5, lacks photosynthetic control and exhibits high ATP synthase conductivity, ruled out loss of these photoprotective mechanisms as causes. Instead, we observed slower formation of proton motive force and by inference ATP synthesis in pgr5 following environmental perturbation, leading to the transient reduction of the electron transfer chain and photosynthetic oscillations. PGR5-dependent CET therefore plays a major role in damping the effect of environmental perturbations on photosynthesis to avoid losses in CO2 fixation.

plant biology↗

bioRxiv2023

Energetic driving force for LHCII clustering in plant membranes

Plants protect themselves against photodamage from excess energy using a process known as non-photochemical quenching (NPQ). A significant fraction of NPQ is induced by a {Delta}pH across the membrane, which changes the conformation, composition, and organization of the antenna complexes. In particular, clustering of the major light-harvesting complex (LHCII) has been observed, yet the thermodynamic driving force behind this reorganization has not been determined, largely because measurements of membrane protein interaction energies have not been possible. Here, we introduce a method to quantify membrane protein interaction energies and its application to the thermodynamics of LHCII clusters. By combining single-molecule measurements of LHCII-proteoliposomes at different protein densities and a rigorous analysis of LHCII clusters and photophysics, we quantified the LHCII-LHCII interaction energy to be approximately -5 kBT at neutral pH and at least -7 kBT at acidic pH. From these values, we found the thermodynamic driving force for LHCII clustering was dominated by these enthalpic contributions. Collectively, this work captures the membrane protein-protein interactions responsible for LHCII clustering from the perspective of equilibrium statistical thermodynamics, which has a long and rich tradition in biology.

biophysics↗

bioRxiv2022

Echolocating bats prefer a high risk-high gain foraging strategy to increase prey profitability

Most bats catch nocturnal prey during active flight guided by echolocation but some species depart from this ancestral behaviour to capture ground prey using passive listening. Here, we explore the costs and benefits of these hunting transitions by combining high-resolution biologging data and DNA metabarcoding to quantify the relative contributions of aerial and ground prey to the total food intake of wild greater mouse-eared bats. We show that these bats use both foraging strategies with similar average nightly captures of 25 small, aerial insects and 30 large, ground-dwelling insects per bat, but with higher capture success in air (78 % in air vs 30 % on ground). However, owing to the 3 to 20 times heavier ground prey, 85 % of the estimated nightly food acquisition comes from ground prey despite the 2.5 times higher failure rates. Further, we find that most bats use the same foraging strategy on a given night suggesting that bats adapt their hunting behaviour to weather and ground conditions. We conclude that prey switching matched to environmental dynamics plays a key role in covering the energy intake even in specialised predators.

animal behavior and cognition↗

bioRxiv2022

Supercharged PGR5-dependent cyclic electron transfer compensates for mis-regulated chloroplast ATP synthase

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 (CBBC). The dominant {Delta}pH component of the pmf also plays a photoprotective role in regulating photosystem II (PSII) light harvesting efficiency, through non-photochemical quenching (NPQ), and cytochrome b6f (cytb6f) to photosystem I (PSI) electron transfer, via photosynthetic control. {Delta}pH 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 {Delta}pH control to photoprotection are not well understood. Here, we show that an Arabidopsis ATP synthase mutant (hope2) with 40% higher proton efflux, has supercharged CET. Double crosses of hope2 with the CET-deficient pgr5 and ndho lines reveal that PGR5-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allows hope2 to maintain wild-type levels of {Delta}pH, CO2 fixation and NPQ, however photosynthetic control remains absent, and PSI is acceptor-side limited. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

plant biology↗

bioRxiv2020

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

plant biology↗

bioRxiv2019

Modelling the role of LHCII-LHCII, PSII-LHCII and PSI-LHCII interactions in state transitions

The light-dependent reactions of photosynthesis take place in the plant chloroplast thylakoid membrane, a complex three-dimensional structure divided into the stacked grana and unstacked stromal lamellae domains. Plants regulate the macro-organization of photosynthetic complexes within the thylakoid membrane to adapt to changing environmental conditions and avoid oxidative stress. One such mechanism is the state transition which regulates photosynthetic light harvesting and electron transfer. State transitions are driven by changes in the phosphorylation of light harvesting antenna complex II (LHCII), which cause a decrease in grana diameter and stacking, a decreased energetic connectivity between photosystem II (PSII) reaction centres and an increase in the relative LHCII antenna size of photosystem I (PSI) compared to PSII. Phosphorylation is believed to drive these changes by weakening the intra-membrane lateral PSII-LHCII and LHCII-LHCII interactions and the inter-membrane stacking interactions between these complexes, while simultaneously increasing the affinity of LHCII for PSI. We investigated the relative roles and contributions of these three types of interaction to state transitions using a lattice-based model of the thylakoid membrane based on existing structural data, developing a novel algorithm to simulate protein complex dynamics. Monte Carlo simulations revealed that state transitions are unlikely to lead to a large-scale migration of LHCII from the grana to the stromal lamellae. Instead, the increased light harvesting capacity of PSI is largely due to the more efficient recruitment of LHCII already residing in the stromal lamellae into PSI-LHCII supercomplexes upon its phosphorylation. Likewise, the increased light harvesting capacity of PSII upon dephosphorylation was found to be driven by a more efficient recruitment of LHCII already residing in the grana into functional PSII-LHCII clusters, primarily driven by lateral interactions. Statement of significanceFor photosynthesis to operate at maximum efficiency the activity of the light-driven chlorophyll-protein complexes, photosystems I and II (PSI and PSII) must be fine-tuned to environmental conditions. Plants achieve this balance through a regulatory mechanism known as the state transition, which modulates the relative light-harvesting antenna size and therefore excitation rate of each photosystem. State transitions are driven by changes in the extent of the phosphorylation of light harvesting complex II (LHCII), which modulate the interactions between PSI, PSII and LHCII. Here we developed a novel algorithm to simulate protein complex dynamics and then ran Monte Carlo simulations to understand how these interactions cooperate to affect the organization of the photosynthetic membrane and bring about state transitions.

plant biology↗