cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

The antagonistic relationship between apoptosis and polyploidy in development and cancer.

One of the important functions of regulated cell death is to prevent cells from inappropriately acquiring extra copies of their genome, a state known as polyploidy. Apoptosis is the primary cell death mechanism that prevents polyploidy, and defects in this apoptotic response can result in polyploid cells whose subsequent error-prone chromosome segregation are a major contributor to genome instability and cancer progression. Conversely, some cells actively repress apoptosis to become polyploid as part of normal development or regeneration. Thus, although apoptosis prevents polyploidy, the polyploid state can actively repress apoptosis. In this review, we discuss progress in understanding the antagonistic relationship between apoptosis and polyploidy in development and cancer. Despite recent advances, a key conclusion is that much remains unknown about the mechanisms that link apoptosis to polyploid cell cycles. We suggest that drawing parallels between the regulation of apoptosis in development and cancer could help to fill this knowledge gap and lead to more effective therapies.

Video-based pooled screening yields improved far-red genetically encoded voltage indicators.

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >103 cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.

De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Cryo-EM structure of the four-subunit Rhodobacter sphaeroides cytochrome bc1 complex in styrene maleic acid nanodiscs.

Cytochrome bc1 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 c1, and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 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 bc1 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 bc1 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 c1 subunits. We observe a quinone at the Qo 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.

Cryo-EM structure of the spinach cytochrome b6 f†complex at 3.6 Šresolution.

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.

Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-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 αß-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 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

STN7 is not essential for developmental acclimation of Arabidopsis to light intensity.

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 b6 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, CO2 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.

Effects of older age on visual and self-motion sensory cue integration in navigation.

Older adults demonstrate impairments in navigation that cannot be explained by general cognitive and motor declines. Previous work has shown that older adults may combine sensory cues during navigation differently than younger adults, though this work has largely been done in dark environments where sensory integration may differ from full-cue environments. Here, we test whether aging adults optimally combine cues from two sensory systems critical for navigation: vision (landmarks) and body-based self-motion cues. Participants completed a homing (triangle completion) task using immersive virtual reality to offer the ability to navigate in a well-lit environment including visibility of the ground plane. An optimal model, based on principles of maximum-likelihood estimation, predicts that precision in homing should increase with multisensory information in a manner consistent with each individual sensory cue's perceived reliability (measured by variability). We found that well-aging adults (with normal or corrected-to-normal sensory acuity and active lifestyles) were more variable and less accurate than younger adults during navigation. Both older and younger adults relied more on their visual systems than a maximum likelihood estimation model would suggest. Overall, younger adults' visual weighting matched the model's predictions whereas older adults showed sub-optimal sensory weighting. In addition, high inter-individual differences were seen in both younger and older adults. These results suggest that older adults do not optimally weight each sensory system when combined during navigation, and that older adults may benefit from interventions that help them recalibrate the combination of visual and self-motion cues for navigation.

Cryo-EM structure of the Blastochloris viridis LH1-RC complex at 2.9 Å.

The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 ß-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between ß-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc 1 complex.

Photosynthesis in the near infrared: the γ subunit of Blastochloris viridis LH1 red-shifts absorption beyond 1000†nm.

The reaction centre (RC) in purple phototrophic bacteria is encircled by the primary light-harvesting complex 1 (LH1) antenna, forming the RC-LH1 'core' complex. The Qy absorption maximum of LH1 complexes ranges from ∼875-960 nm in bacteriochlorophyll (BChl) a-utilising organisms, to 1018 nm in the BChl b-containing complex from Blastochloris (Blc.) viridis. The red-shifted absorption of the Blc. viridis LH1 was predicted to be due in part to the presence of the γ subunit unique to Blastochloris spp., which binds to the exterior of the complex and is proposed to increase packing and excitonic coupling of the BChl pigments. The study by Namoon et al. provides experimental evidence for the red-shifting role of the γ subunit and an evolutionary rationale for its incorporation into LH1. The authors show that cells producing RC-LH1 lacking the γ subunit absorb maximally at 972 nm, 46 nm to the blue of the wild-type organism. Wavelengths in the 900-1000 nm region of the solar spectrum transmit poorly through water, thus γ shifts absorption of LH1 to a region where photons have lower energy but are more abundant. Complementation of the mutant with a divergent copy of LH1γ resulted in an intermediate red shift, revealing the possibility of tuning LH1 absorption using engineered variants of this subunit. These findings provide new insights into photosynthesis in the lowest energy phototrophs and how the absorption properties of light-harvesting complexes are modified by the recruitment of additional subunits.

Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

Impact of Appropriate Use Criteria for Cross-Sectional Imaging on Medical Decision-Making in Patients with Conotruncal Defects.

Associations between Appropriate Use Criteria (AUC) ratings and medical decision-making in congenital heart disease are not well-established. We applied the 2020 AUC for multimodality imaging in follow-up care of pediatric and young adult patients with conotruncal defects to evaluate appropriateness of cardiac magnetic resonance (CMR) and computed tomography (CCT) use in this population and impact on clinical decision-making. Records were reviewed and assigned AUC indications and corresponding ratings for CMR and CCT. We examined the relationship between AUC indications, their ratings, and change in management. Of the 200 studies (133 CMR, 67 CCT) performed on 187 patients, no studies were rated Rarely Appropriate (R), and most studies were obtained for routine follow-up (151/200 [75.5%]) and were not prompted by clinical concerns. There were 70/200 (35.0%) studies which led to management changes; these included transcatheter intervention (29/70 [41.4%]), surgical intervention (25/70 [35.7%]), other interventions (10/70 [14.3%]), and medical intervention (6/70 [8.6%]). Among all studies, studies prompted by clinical concerns and studies rated M more frequently resulted in change in management (46.9 vs 31.1%, p = 0.04 and 54.1 vs 30.7%, p = 0.003, respectively). In conclusion, we found that all studies were ordered for indications rated Appropriate (A) or May be Appropriate (M), indicating compliance in ordering practices as outlined by published AUC. Studies ordered for clinical change or rated M more frequently led to management change in patient care. Findings may help inform provider expectations of testing yield in this population and serve as a platform for development of future iterations of AUC.

Contemporary transesophageal echocardiography practice patterns among paediatric cardiology centres in the United States and Canada.

OBJECTIVE: To characterise transesophageal echocardiography practice patterns among paediatric cardiac surgical centres in the United States and Canada. METHODS: A 42-question survey was sent to 80 echocardiography laboratory directors at paediatric cardiology centres with surgical programmes in the United States and Canada. Question domains included transesophageal echocardiography centre characteristics, performance and reporting, equipment use, trainee participation, and quality assurance. RESULTS: Fifty of the 80 centres (62.5%) responded to the survey. Most settings were academic (86.0%) with 42.0% of centres performing > 350 surgical cases/year. The median number of transesophageal echocardiograms performed/cardiologist/year was 50 (26, 73). Pre-operative transesophageal echocardiography was performed in most surgical cases by 91.7% of centres. Transesophageal echocardiography was always performed by most centres following Norwood, Glenn, and Fontan procedures and by < 10% of centres following coarctation repair. Many centres with a written guideline allowed transesophageal echocardiography transducer use at weights below manufacturer recommendations (50.0 and 61.1% for neonatal and paediatric transducers, respectively). Most centres (36/37, 97.3%) with categorical fellowships had rotations which included transesophageal echocardiography participation. Large surgical centres (>350 cases/year) had higher median number of transesophageal echocardiograms/cardiologist/year (75.5 [53, 86] versus 35 [20, 52], p < 0.001) and more frequently used anaesthesia for diagnostic transesophageal echocardiography ≥ 67% of time (100.0 versus 62.1%, p = 0.001). CONCLUSIONS: There is significant variability in transesophageal echocardiography practice patterns and training requirements among paediatric cardiology centres in the United States and Canada. Findings may help inform programmatic decisions regarding transesophageal echocardiography expectations, performance and reporting, equipment use, trainee involvement, and quality assurance.

Comparison of post-operative transesophageal and transthoracic echocardiogram findings following atrioventricular septal defect repair.

Success of atrioventricular septal defect repair is defined by post-operative atrioventricular valve function and presence of residual intracardiac shunting. We evaluated differences in interpretation of atrioventricular valve function and residual defects between transesophageal and transthoracic echocardiography in a contemporary cohort of infants undergoing atrioventricular septal defect repair. Among 106 patients, we identified an increase in left and right atrioventricular valve regurgitation, right atrioventricular valve inflow gradient, and increased detection rate of residual intracardiac shunting on transthoracic compared to transesophageal echocardiograms, although residual shunts identified only on transthoracic echocardiogram were not haemodynamically significant. Findings may help inform expectation of post-operative transthoracic echocardiogram findings based on intraoperative assessment.

Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICEBs1, an integrative and conjugative element in Bacillus subtilis. ConE, encoded by ICEBs1, is a member of the VirB4 family of ATPases, the most conserved component of T4SSs. ConE is required for conjugation and localizes to the cell membrane, predominantly at the cell poles. In addition to Walker A and B boxes, VirB4 homologs have conserved ATPase motifs C, D, and E. Here, we created alanine substitutions in five conserved residues within or near ATPase motifs in ConE. Mutations in all five residues drastically decreased conjugation frequency but did not affect ConE protein levels or localization, indicating that an intact ATPase domain is critical for DNA transfer. Purified ConE is largely monomeric with some oligomers and lacks enzymatic activity, suggesting that ATP hydrolysis may be regulated or require special solution conditions. Finally, we investigated which ICEBs1 T4SS components interact with ConE using a bacterial two-hybrid assay. ConE interacts with itself, ConB, and ConQ, but these interactions are not required to stabilize ConE protein levels and largely do not depend on conserved residues within the ATPase motifs of ConE. The structure-function characterization of ConE provides more insight into this conserved component shared by all T4SSs. IMPORTANCE Conjugation is a major form of horizontal gene transfer and involves the transfer of DNA from one bacterium to another through the conjugation machinery. Conjugation contributes to bacterial evolution by disseminating genes involved in antibiotic resistance, metabolism, and virulence. Here, we characterized ConE, a protein component of the conjugation machinery of the conjugative element ICEBs1 of the bacterium Bacillus subtilis. We found that mutations in the conserved ATPase motifs of ConE disrupt mating but do not alter ConE localization, self-interaction, or levels. We also explored which conjugation proteins ConE interacts with and whether these interactions contribute to stabilizing ConE. Our work contributes to the understanding of the conjugative machinery of Gram-positive bacteria.

Phycobilisome's Exciton Transfer Efficiency Relies on an Energetic Funnel Driven by Chromophore-Linker Protein Interactions.

The phycobilisome is the primary light-harvesting antenna in cyanobacterial and red algal oxygenic photosynthesis. It maintains near-unity efficiency of energy transfer to reaction centers despite relying on slow exciton hopping along a relatively sparse network of highly fluorescent phycobilin chromophores. How the complex maintains this high efficiency remains unexplained. Using a two-dimensional electronic spectroscopy polarization scheme that enhances energy transfer features, we directly watch energy flow in the phycobilisome complex of Synechocystis sp. PCC 6803 from the outer phycocyanin rods to the allophycocyanin core. The observed downhill flow of energy, previously hidden within congested spectra, is faster than timescales predicted by Förster hopping along single rod chromophores. We attribute the fast, 8 ps energy transfer to interactions between rod-core linker proteins and terminal rod chromophores, which facilitate unidirectionally downhill energy flow to the core. This mechanism drives the high energy transfer efficiency in the phycobilisome and suggests that linker protein-chromophore interactions have likely evolved to shape its energetic landscape.