Proceedings of the National Academy of Sciences of the United States of America最新文献

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In Escherichia coli, the α-amino-linked lipid of lipoproteins is added by the lipoprotein N-acyltransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides, named lipoprotein N-acyltransferase in Bacteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in gram-negative bacteria but also suggest the existence of a new family of lipoprotein N-acyltransferases.

There has been recent interest in trying to understand the connection between transfer RNA (tRNA) posttranscriptional modifications and changes in-cellular environmental conditions. Here, we report on the identification of the modified nucleoside 5-methylcytidine (m⁵C) in Escherichia coli tRNAs. This modification was determined to be present at position 49 of tRNA Tyr-QUA-II. Moreover, m⁵C levels in this tRNA are significantly elevated under high reactive oxygen specieis (ROS) conditions in E. coli cells. We identified the known ribosomal RNA methyltransferase rsmF as the enzyme responsible for m⁵C synthesis in tRNA and enzyme transcript levels are responsive to elevated levels of ROS in the cell. We further find that changes in m⁵C levels in this tRNA are not specific to Fenton-like reaction conditions elevating ROS, but heat shock can also induce increased modification of tRNA Tyr-QUA-II. Altogether, this work illustrates how cells adapt to changing environmental conditions through variations in tRNA modification profiles.

Movies are a massively popular and influential form of media, but their computational study at scale has largely been off-limits to researchers in the United States due to the Digital Millennium Copyright Act. In this work, we illustrate use of a new regulatory framework to enable computational research on film that permits circumvention of technological protection measures on digital video discs (DVDs). We use this exemption to legally digitize a collection of 2,307 films representing the top 50 movies by U.S. box office over the period 1980 to 2022, along with award nominees. We design a computational pipeline for measuring the representation of gender and race/ethnicity in film, drawing on computer vision models for recognizing actors and human perceptions of gender and race/ethnicity. Doing so allows us to learn substantive facts about representation and diversity in Hollywood over this period, confirming earlier studies that see an increase in diversity over the past decade, while allowing us to use computational methods to uncover a range of ad hoc analytical findings. Our work illustrates the affordances of the data-driven analysis of film at a large scale.

To address rising global food demand, the development of sustainable technologies to increase productivity is urgently needed. This study revealed that foliar application of zinc oxide nanoparticles (ZnO NPs; 30 to 80 nm, 0.67 mg/d per plant, 6 d) to rice leaves under heatwave (HW) stress increased the grain yield and nutritional quality. Compared with the HW control, the HWs+ZnO group presented increases in the grain yield, grain protein content, and amino acid content of 22.1%, 11.8%, and 77.5%, respectively. Nanoscale ZnO aggregated on the leaf surface and interacted with leaf surface molecules. Compared with that at ambient temperature, HW treatment increased the dissolution of ZnO NPs on the leaf surface by 25.9% and facilitated their translocation to mesophyll cells. The Zn in the leaves existed as both ionic Zn and particulate ZnO. Compared with the HW control, foliar application of ZnO NPs under HW conditions increased leaf nutrient levels (Zn, Mn, Cu, Fe, and Mg) by 15.8 to 416.9%, the chlorophyll content by 22.2 to 24.8%, Rubisco enzyme activity by 21.2%, and antioxidant activity by 26.7 to 31.2%. Transcriptomic analyses revealed that ZnO NPs reversed HW-induced transcriptomic dysregulation, thereby enhancing leaf photosynthesis by 74.4%. Additionally, ZnO NPs increased the diversity, stability, and enrichment of beneficial microbial taxa and protected the phyllosphere microbial community from HW damage. This work elucidates how NPs interact with the phyllosphere, highlighting the potential of NPs to promote sustainable agriculture, especially under extreme climate events (e.g., HWs).

Oysters (Ostreidae) play a pivotal role in the health and productivity of marine ecosystems. Their unique ability to filter water, provide habitat, and contribute to nutrient cycling has remained underused in many parts of Europe following the destruction of vast oyster beds in the 19th and 20th centuries. The burgeoning field of oyster restoration for aquaculture has recognized the potential of these bivalves in promoting ecosystem resilience and enhancing biodiversity. Restoring oysters to previous levels requires the establishment of ecological baselines that ideally take into account the long-term changes of animal behavior as well as the surrounding environment prior to significant human intervention, an extremely challenging task. Archaeological shell middens are invaluable baseline archives and provide exclusive insights into past ecosystems. Here, we use demographic information from over 2,000 analyzed European oyster (Ostrea edulis) shells dating from ~5,660 to 2,600 cal BCE (calibrated years BCE), the largest archaeological growth rate dataset of mollusks yet. Through the analysis of size as well as ontogenetic age, we decouple anthropogenic from environmental impacts throughout Denmark. Our data show definitive influence of oyster size-age structure through human harvesting during the Mid-Holocene, with older oysters in the Mesolithic (mean: 4.9 y) than the Neolithic (mean: 3.7 y), irrespective of changes in growth rate. Furthermore, we present the metrics for long-term sustainable harvesting of oysters across environmental and socioeconomic transitions, providing demographic targets for current oyster restoration projects and valuable context in mitigating the impact of modern climatic change.

Whole-genome sequencing of bacterial pathogens is used by public health agencies to link cases of food poisoning caused by the same source of contamination. The vast majority of these appear to be sporadic cases associated with small contamination episodes and do not trigger investigations. A "contamination episode" refers to one or more contamination events from a single source over a period of time. We examine clusters of sequenced clinical isolates of Salmonella, Escherichia coli, Campylobacter, and Listeria that differ by only a small number of mutations (SNPs) to identify features of the underlying contamination episodes. These analyses provide additional evidence that the youngest age groups have greater susceptibility to infection by Salmonella, E. coli, and Campylobacter than older age groups. This age bias is weaker for the common Salmonella serovar Enteritidis than Salmonella in general. A large fraction of the contamination episodes causing sickness appear to have a long duration. For example, 50% of the Salmonella cases are in clusters that persist for almost 3 y. For all four pathogen species, the majority of the cases were part of genetic clusters with illnesses in multiple states and likely to be caused by contaminated commercially distributed foods. Salmonella infections in infants under 3 mo are predominantly acquired from the same contaminated food, pet food, or environmental sources as older individuals, rather than infant formula contaminated during production.

To maintain CO₂ fixation in the Calvin-Benson-Bassham cycle, multistep regulation of the chloroplast ATP synthase (CF₁F_o) is crucial to balance the ATP output of photosynthesis with protection of the apparatus. A well-studied mechanism is thiol modulation; a light/dark regulation through reversible cleavage of a disulfide in the CF₁F_o γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF₁F_o from land plants by increasing the required transmembrane electrochemical proton gradient ([Formula: see text]). Here, we show in Chlamydomonas reinhardtii that algal CF₁F_o is differently regulated in vivo. A specific hairpin structure in the γ-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild-type CF₁F_o active, whereas the enzyme was switched off in algal mutant cells expressing a plant-like hairpin structure. The hairpin segment swap resulted in an elevated [Formula: see text] threshold to activate plant-like CF₁F_o, increased by ~1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula: see text] dropped in the mutants by ~2.7 PSI charge separation equivalents. Photobioreactor experiments showed no phenotypes in autotrophic aerated mutant cultures. In contrast, chlorophyll fluorescence measurements under heterotrophic dark conditions point to an altered dark metabolism in cells with the plant-like CF₁F_o as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of C. reinhardtii requires a specific CF₁F_o dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria.

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 variant, R256H. We previously identified this variant in a family with dilated cardiomyopathy, who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using cryoelectron microscopy, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1^R256H/+ human-induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric organization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.

The proton-motive force (PMF), consisting of a pH gradient and a membrane potential (ΔΨ) underpins many processes essential to bacterial growth and/or survival. Yet bacteria often enter a bioenergetically diminished state characterized by a low PMF. Consequently, they have increased tolerance for diverse stressors, including clinical antibiotics. Despite the ubiquity of low metabolic rates in the environment, the extent to which bacteria have agency over entry into such a low-bioenergetic state has received relatively little attention. Here, we tested the hypothesis that production of redox-active metabolites (RAMs) could drive such a physiological transition. Pseudomonas aeruginosa is an opportunistic pathogen that produces phenazines, model RAMs that are highly toxic in the presence of molecular oxygen (O₂). Under oxic conditions, the phenazines pyocyanin and phenazine-1-carboximide, as well as toxoflavin-a RAM produced by Burkholderia species-suppress the ΔΨ in distinct ways across distributions of single cells, reduce the efficiency of proton pumping, and lower cellular adenosine-triphosphate (ATP) levels. In planktonic culture, the degree and rate by which each RAM lowers the ΔΨ correlates with the protection it confers against antibiotics that strongly impact cellular energy flux. This bioenergetic suppression requires the RAM's presence and corresponds to its cellular reduction rate and abiotic oxidation rate by O₂; it can be reversed by increasing the ΔΨ with nigericin. RAMs similarly impact the bioenergetic state of cells in (hyp)oxic biofilm aggregates. Collectively, these findings demonstrate that bacteria can suppress their bioenergetic state by the production of endogenous toxins in a manner that bolsters stress resilience.

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type 6 secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a ΔhapR strain displays no cell death at the colony rim. RNA-Sequencing (RNA-Seq) analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647, are required to drive cell death in both a ΔhapR and a ΔhapR Δt6ss strain. Thus, vca0646-0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae.