Information theoretic approaches are ubiquitous and effective in a wide variety of bioinformatics applications. In comparative genomics, alignment-free methods, based on short DNA words, or k-mers, ...are particularly powerful. We evaluated the utility of varying k-mer lengths for genome comparisons by analyzing their sequence space coverage of 5805 genomes in the KEGG GENOME database. In subsequent analyses on four k-mer lengths spanning the relevant range (11, 21, 31, 41), hierarchical clustering of 1634 genus-level representative genomes using pairwise 21- and 31-mer Jaccard similarities best recapitulated a phylogenetic/taxonomic tree of life with clear boundaries for superkingdom domains and high subtree similarity for named taxons at lower levels (family through phylum). By analyzing ~14.2M prokaryotic genome comparisons by their lowest-common-ancestor taxon levels, we detected many potential misclassification errors in a curated database, further demonstrating the need for wide-scale adoption of quantitative taxonomic classifications based on whole-genome similarity.
Summary
The process of oxygenic photosynthesis enabled and still sustains aerobic life on Earth. The most elaborate form of the apparatus that carries out the primary steps of this vital process is ...the one present in higher plants. Here, we review the overall composition and supramolecular organization of this apparatus, as well as the complex architecture of the lamellar system within which it is harbored. Along the way, we refer to the genetic, biochemical, spectroscopic and, in particular, microscopic studies that have been employed to elucidate the structure and working of this remarkable molecular energy conversion device. As an example of the highly dynamic nature of the apparatus, we discuss the molecular and structural events that enable it to maintain high photosynthetic yields under fluctuating light conditions. We conclude the review with a summary of the hypotheses made over the years about the driving forces that underlie the partition of the lamellar system of higher plants and certain green algae into appressed and non‐appressed membrane domains and the segregation of the photosynthetic protein complexes within these domains.
Plant photosynthetic (thylakoid) membranes are organized into complex networks that are differentiated into 2 distinct morphological and functional domains called grana and stroma lamellae. How the 2 ...domains join to form a continuous lamellar system has been the subject of numerous studies since the mid-1950s. Using different electron tomography techniques, we found that the grana and stroma lamellae are connected by an array of pitch-balanced right- and left-handed helical membrane surfaces of different radii and pitch. Consistent with theoretical predictions, this arrangement is shown to minimize the surface and bending energies of the membranes. Related configurations were proposed to be present in the rough endoplasmic reticulum and in dense nuclear matter phases theorized to exist in neutron star crusts, where the right- and left-handed helical elements differ only in their handedness. Pitch-balanced helical elements of alternating handedness may thus constitute a fundamental geometry for the efficient packing of connected layers or sheets.
The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into ...two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.
Cellular lineage tracking provides a means to observe population makeup at the clonal level, allowing exploration of heterogeneity, evolutionary and developmental processes and individual clones' ...relative fitness. It has thus contributed significantly to understanding microbial evolution, organ differentiation and cancer heterogeneity, among others. Its use, however, is limited because existing methods are highly specific, expensive, labour-intensive, and, critically, do not allow the repetition of experiments. To address these issues, we developed gUMI-BEAR (genomic Unique Molecular Identifier Barcoded Enriched Associated Regions), a modular, cost-effective method for tracking populations at high resolution. We first demonstrate the system's application and resolution by applying it to track tens of thousands of Saccharomyces cerevisiae lineages growing together under varying environmental conditions applied across multiple generations, revealing fitness differences and lineage-specific adaptations. Then, we demonstrate how gUMI-BEAR can be used to perform parallel screening of a huge number of randomly generated variants of the Hsp82 gene. We further show how our method allows isolation of variants, even if their frequency in the population is low, thus enabling unsupervised identification of modifications that lead to a behaviour of interest.
Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not ...been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms.
Significance The fitness and robustness of plants crucially depend on the molecular repair of the vulnerable photosystem II (PS II) supercomplex, embedded in photosynthetic thylakoid membranes. To ...maintain photosynthetic performance, plants evolved an efficient multistep PS II repair cycle. The PS II repair cycle relies on a well-defined order of reactions and partial separation of individual repair steps. By combining biochemical, spectroscopic, and ultrastructural techniques, we discover that plants establish reaction order and separation by confinement of the enzymes that catalyze the individual steps to spatially separated thylakoid subcompartments—grana, grana margins, and stroma lamellae—formed by the stacked membranes. Structural flexibility of the thylakoid architecture allows controlled access of the damaged PS II by the repair machinery.
A crucial component of protein homeostasis in cells is the repair of damaged proteins. The repair of oxygen-evolving photosystem II (PS II) supercomplexes in plant chloroplasts is a prime example of a very efficient repair process that evolved in response to the high vulnerability of PS II to photooxidative damage, exacerbated by high-light (HL) stress. Significant progress in recent years has unraveled individual components and steps that constitute the PS II repair machinery, which is embedded in the thylakoid membrane system inside chloroplasts. However, an open question is how a certain order of these repair steps is established and how unwanted back-reactions that jeopardize the repair efficiency are avoided. Here, we report that spatial separation of key enzymes involved in PS II repair is realized by subcompartmentalization of the thylakoid membrane, accomplished by the formation of stacked grana membranes. The spatial segregation of kinases, phosphatases, proteases, and ribosomes ensures a certain order of events with minimal mutual interference. The margins of the grana turn out to be the site of protein degradation, well separated from active PS II in grana core and de novo protein synthesis in unstacked stroma lamellae. Furthermore, HL induces a partial conversion of stacked grana core to grana margin, which leads to a controlled access of proteases to PS II. Our study suggests that the origin of grana in evolution ensures high repair efficiency, which is essential for PS II homeostasis.
Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and ...computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.
Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants ...and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.
Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition ...takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.
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