Biology Direct

Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and non-coding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in one condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive dataset assigns condition-specific importance to many previously unannotated genes, and suggests new functions for central metabolic enzymes. Prk, GAPDH, and CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion in the first 12 bp proximal to the PAM site. Fitness data for all genes in all conditions is compiled in an interactive web application.

Circadian rhythms orchestrate gene expression to align plant growth and development with daily environmental cycles. However, the post-transcriptional mechanisms that coordinate transcriptional and translational rhythmicity remain incompletely understood. To address this, we analyzed time-series transcriptome and translatome profiles in Arabidopsis seedlings, identifying 5,185 genes with rhythmicity at both levels. These genes were classified into four distinct groups based on phase and amplitude differences between transcription and translation. Circadian mRNAs with high oscillation amplitudes tended to undergo co-translational RNA decay (CTRD), whereas intronless genes displayed the lowest amplitudes, likely due to their mRNA instability and short half-lives. While CTRD and NAD capping modulate amplitude differences, intronless and circadian translational efficiency (TE) influence both phase and amplitude variations. Additionally, CTRD, NAD+ capping and circadian TE facilitate fast recovery of heat-induced genes to normal hemostasias. Collectively, our findings demonstrate that these post-transcriptional regulation shapes both synchronized and decoupled transcription and translation during plants response to diel and environmental dynamics.

Phage-plasmids (PPs), hybrid mobile genetic elements possessing characteristics of both plasmids and bacteriophages, have recently gained attention for their proposed role in horizontal gene transfer, particularly in the dissemination of antimicrobial resistance genes (ARGs). In this study, we re-evaluated the prevalence, structure, and functional attributes of PPs across publicly available datasets comprising over 5 million viral and plasmid sequences. By employing a conservative and domain-centric approach that strictly filtered for high-quality phage genomes devoid of insertion sequences (ISs) and containing plasmid hallmark domains, we identified 3,002 putative PPs--representing a significantly lower proportion ([~]0.25%) than previous estimates. Our functional analyses revealed that PPs are more closely related to virulent phages than to temperate phages or plasmids and primarily encode partitioning proteins rather than conjugative machinery, suggesting episomal maintenance and vertical inheritance. PPs displayed genome sizes larger than most phages or plasmids, indicating a potential fitness cost to their bacterial hosts and explaining their rarity. Despite prior claims, we found no evidence of ARG carriage in PPs or virulent phages; only a minority of temperate phages harbored such genes. Furthermore, the dihydrofolate reductase genes commonly mistaken as ARGs were excluded due to their structural and functional roles during phage infection. Interestingly, majority of the PPs were Caudoviricetes with plasmid partitioning proteins, and PPs classified under Faserviricetes universally carried the relaxase NicK, likely reflecting their rolling-circle replication rather than conjugative potential. Our findings challenge earlier generalizations regarding PPs and support a revised view that emphasizes their derivation from bacteriophages, limited functional resemblance to plasmids, and limited role in ARG dissemination.

Levacetylleucine (AqneursaTM), a chemically modified amino acid, is the only US Food and Drug Administration-approved monotherapy for the treatment of Niemann-Pick disease type C (NPC) (Beninger, 2024; Mullard, 2024; van Gool et al., 2025). This acetylated derivative of L-leucine functions as a pro-drug, with the acetyl group rendering it a substrate for the monocarboxylate transporter (MCT) family of transporters to allow appreciable penetration of the blood-brain barrier and its efficient uptake into cells (Churchill et al., 2021). Inside cells, levacetylleucine undergoes metabolism catalysed by acylases, and the resultant high quantities of L-leucine enter metabolic pathways which enhance mitochondrial bioenergetics and, as previously demonstrated, indirectly ameliorate lysosomal function (Kaya et al., 2020). Here, we show a novel aspect of levacetylleucines mechanism of action, demonstrating a direct effect on lysosomal function through its rapid modulation of the translocation of the transcription factor TFEB, a master regulator of lysosomal biogenic and autophagic genes (Napolitano and Ballabio, 2016), from cytoplasm to nucleus. Uniquely, we have demonstrated a biphasic action whereby levacetylleucine normalizes TFEB activity, consistent with levacetylleucines previously shown ability to regulate cellular homeostasis: in wild-type HeLa cells, levacetylleucine enhances and activates the translocation of TFEB to the nucleus. In contrast, in cellular models of NPC type 1 disease, where TFEB is already over-expressed in the nucleus (as the cell attempts to compensate for the primary defect by activating TFEB as a natural cellular response to the lysosomal substrate accumulation and associated cellular stress), treatment with levacetylleucine down-regulates and restores the distribution of TFEB to a more normalized cytoplasmic: nuclear ratio. Importantly, both effects of levacetylleucine occur at concentrations consistent with plasma concentrations in therapeutic dosing (Churchill et al., 2020). The effects were also confirmed to be stereospecific to the L-enantiomer, as neither the D-enantiomer (N-acetyl-D-leucine) or racemate (N-acetyl-DL-leucine) had any effect, The presence of the D-enantiomer in the racemic mixture inhibited the ability of levacetylleucine to promote TFEB bidirectional translocation, consistent with previous studies, which have established antagonism of N-acetyl-L-leucine by N-acetyl-D-leucine in the racemic mixture (rendering the racemic mixture without effect). This bidirectional mechanism of action of levacetylleucine to impact lysosomal function directly and normalize, either by activating basal TFEB signalling or reducing aberrant TFEB function in NPC1 knockout cells, thereby modulating lysosomal and autophagic functions, lends itself to the treatment of a broad range of neurological and neurodevelopment disorders.

The gut microbiome has been implicated as a major factor contributing to metabolic diseases as well as being contributors to the response to drugs used for the treatment of such diseases. In this study, using a diet-induced obesity mouse model, we tested the effect of cholestyramine, a bile acid sequestrant, on the murine gut microbiome and mammalian metabolism. We also explored the hypothesis that some beneficial effects of this drug on systemic metabolism can be attributed to alterations in gut microbiota. First, we demonstrated that cholestyramine can decrease glucose and epidydimal fat levels. Next, while investigating gut microbiota we found increased alpha diversity of the gut microbiome of cholestyramine-treated mice, with fourteen taxa showing restoration of abundance to levels resembling those in mice fed with a control diet. Analyzing expression of genes known to be regulated by cholestyramine (including Cyp7a1), we confirmed the expected effect of this drug in the liver and ileum. Finally, using a transkingdom network analysis we inferred Acetatifactor muris and Muribaculum intestinale as potential mediators/modifiers of cholestyramine effects on the mammalian host. In addition, A. muris correlated positively with glucagon (Gcg) expression in the ileum and negatively correlated with small heterodimer partner (Shp) expression in the liver. Interestingly, A. muris also correlated negatively with glucose levels, further indicating the potential probiotic role for A. muris. In conclusion, our results indicate the gut microbiome has a role in the beneficial effects of cholestyramine and suggest specific microbes as targets of future investigations.

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid". Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

Complex regulatory interactions between genetic and metabolic networks together confer robustness against external and internal perturbations in an organism such as Escherichia coli. In balanced exponential growth, this robustness is attributed to cost-effective metabolism by means of efficient resource allocation coordinated by the interplay of global transcriptional regulators with growth-rate dependent machinery. Here, we reappraise the role of global transcriptional regulators FNR, ArcA and IHF, integral to sustaining proteome-efficiency in anaerobic fermentative conditions, fundamental for optimal growth of E. coli. We reveal at the transcriptome and metabolome level, that absence of these global regulators ensued a disruption of nitrogen homeostasis, overexpression of otherwise unnecessary or hedging genes and impairment in core bottleneck steps and amino acid metabolism. Notably, our findings emphasize their importance in optimizing the metabolic proteome resources essential for rapid exponential growth. Consequentially, the perturbations in the metabolic proteome as a result of deletion of global regulators unbalances the ribosomal proteome share imposing a high translation program, though at the expense of lowered efficiency. We illustrate that disruption of this inherent trade-off between metabolic and ribosomal proteomic investment eventually culminate to lowered growth rates. Despite no changes in gene expression related to glucose import, our findings elucidate that the accumulations of intracellular metabolites directly modulated by growth rate, negatively impacts the glucose uptake. Our results employing the proteome allocation theory and quantitative experimental measurements, suffices to explain the physiological consequences of altered translational and metabolic efficiency in the cell, driven by the loss of these global regulators.

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including three AGOs and three Dicer-like (DCL) proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long sRNAs (>26 nt), which associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these long sRNAs are derived from moderately repetitive genomic clusters and their biogenesis appears to be Dicer-independent. Interestingly, long sRNA encoding sequences have been conserved and amplified in phylogenetically related Chlamydomonas species. Additionally, expression of several long sRNAs increases substantially under nutrient deprivation, correlating with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences encoding long sRNAs might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, some long sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. {Delta}amt mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. The analytical data highlights several internal adaptations, including increased N assimilation (i.e. greater GS activity) and nitrogenase activity (i.e. faster activation upon N deprivation) together with altered amino acids metabolism, as indicated by changes in Gln, Glu and 2-OG, indicating an altered C/N balance. The analyses provides evidence for an active role of AMT transporters in the regulatory/signalling network of N metabolism in this biological system, and the existence of a novel fourth IF7A-independent regulatory mechanism controlling GS activity.

Gene expression is time-consuming and sequentially more so from bacteria to yeast to human, rendering human cells vulnerable to proteomic-response and operational latency. Computers once suffered such latency, imposed by much-slower information retrieval (hard-drive (HD) to memory to central-processing-unit (CPU)) than CPU execution. Optimization principles, namely, spatiotemporal-locality-principles that control specialized user-programs and caching that controls operating system (OS) kernel (the HD-CPU information flow channel), successfully mitigated the latency by gearing the memory towards near-future or high-priority CPU needs. We report evidence that the principles similarly act in cellular latency-mitigation via analogizing genome-mRNA-protein gene-expression to HD-memory-CPU information-retrieval, and transcriptome to memory. First, temporal-locality-principle is equivalent to mRNA stabilization-by-translation regulation and controls specialized cellular functions. Second, caching is equivalent to cytoplasmic mRNA sequestration. Highly sequestered mRNAs defy the locality-principle. In both cells and computers, caching controls the information channels; gene expression machinery and their regulators, i.e., the cellular channel (OS-kernel equivalent) that regulates arguably all cellular processes, are top sequestered mRNAs. Third, mRNA-caching contributes to the mRNA-protein expression discrepancy. Thus, locality and caching principles control specialized and core cellular functions, respectively, orchestrating transcriptome regulation and bridging it to cellular operational efficiency.

Pyridoxal-phosphate binding proteins (PLPBP) are involved in the homeostasis of B6 vitamers and amino/keto acids, share a high degree of sequence conservation and are represented in all three domains of life. Despite the obligate presence of the catalyst cofactor PLP, attempts to show enzymatic activity have been unsuccessful. Instead, evidence of RNA binding activity has been provided for several members of the family. Here we use PipY, one of the few PLBPB members studied so far, as a model system to address the phenotypic impact in the cyanobacterium Synechococcus elongatus of mutations K26A, P63L and R210Q, which respectively prevent PLP binding or are equivalent to those conferring B6-dependent epilepsy in humans with a recessive inheritance pattern. We found that while mutation K26A at the PLP-binding residue abrogated all phenotypes associated to PipY overexpression and toxicity, P63L and R210Q behaved as dominant gain-of-function mutations that inhibited bacterial growth. We provide in vivo evidence of PipY performing PLP-independent functions, in which mutant variant PipYK26A but not PipYP63L or PipYR210Q would be defective. A model integrating our observations with previous data from other organims and PLPBP variants is discussed.

Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness cost HighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal medium {Delta}4 rrn and rpoB mutants share phenotypic traits Faster growth of mutants is achieved by increased ribosome content Fast-growing mutants display reduced hedging expression and adaptation trade-offs Despite similar ribosomal content in rich medium the mutants present growth defects

We discovered an enhanced functionality hammerhead ribozyme (EhhRz), designed to act in trans against human rod opsin (RHO) mRNA, with turnover activity >300 nM min-1 under substrate-excess conditions and physiological Mg2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC{downarrow} cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess (Vmax/Km up to 1.60 x 108 min-1 M-1), which is comparable to RNase A. EhhRzs show cooperative titration with a Kd of 0.73 {+/-} 0.02 mM at cellular Mg2+ concentrations and a Hill coefficient of 1.73 {+/-} 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min-1. These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic.

Radiation-resistant Deinococcus radiodurans is an extremophilic microorganism capable of withstanding high levels of ionizing radiation and chemical mutagens. It possesses remarkable DNA repair capability and serves as a model organism for studying stress resistance mechanism. However, our understanding on the spatial chromosome organization of this species remains limited. In this study, we employed chromosome conformation capture (3C) technology to determine the 3D genome structure of D. radiodurans and to further investigate the changes of chromosome conformation induced by ultraviolet (UV) irradiation. We observed that UV irradiation reduced short-range chromosome interactions, and smaller chromosomal interaction domains (CIDs) merged to form larger CIDs. Integrating transcriptomic data analysis, we found that the majority of upregulated differentially expressed genes were significantly enriched near specific CID boundaries. Specially, we comprehensively elucidated that the nucleoid-associated protein Dr_ebfC may serve as a global regulator for gene expression by altering chromosome structure, thereby influencing the physiological state of the bacterium. Overall, our study revealed the chromosome conformations of D. radiodurans under different conditions, and offered valuable insights into the molecular responses of this extremophile to environmental stresses.

The molecular mechanisms for repairing DNA damages and point mutations have been well understood but it remains unclear how a frameshift mutation is repaired. Here we report that frameshift reversion occurs in E. coli more frequently than expected and appears to be a targeted gene repair signaled by premature termination codons (PTCs), producing high-level variations in the repaired genes. Genome resequencing shows that the revertant genome is highly stable, and the single-molecule variations in the repaired genes are derived from RNA editing. A multi-omics analysis shows that the expression levels change greatly in most the DNA and RNA manipulating genes. DNA replication, transcription, RNA editing, RNA degradation, nucleotide excision repair, mismatch repair, and homologous recombination were upregulated in the frameshift or revertant, but the base excision repair was not. Moreover, genes and transposons in a duplicate region silenced in wild type E. coli were activated in the frameshift. Finally, we propose a nonsense-mediated gene revising (NMGR) model for frame repair, which also acts as a driving force for molecular evolution. In essence, nonsense mRNAs are recognized, edited, and transported to template the repair of the coding gene by RNA-directed DNA repair, nucleotide excision, mismatch repair, and homologous recombination. Thanks to NMGR, the mutation rate temporarily rises in a frameshift gene, bringing genetic diversity while repairing the frameshift mutation and accelerating the evolution process without a high mutation rate in the genome.

Thylakoid membranes (TM) in cyanobacteria and chloroplasts host the light-dependent reactions of oxygenic photosynthesis, which involve a linear electron transfer (LET) chain composed of multi-subunit complexes, including notably Photosystem II (PSII). Gloeobacterales, the earliest-diverging cyanobacterial lineage, lack TM and perform photosynthesis within specialized regions of the cytoplasmic membrane (CM), thereby representing an ancestral state with respect to other cyanobacteria, all equipped with TM and known as Phycobacteria. The emergence of TM, which increased the membrane surface available for oxygenic photosynthesis, was a key innovation that likely contributed to the Great Oxidation Event. This evolutionary transition involved the formation of a distinct membrane compartment, followed by the relocation of LET components from the CM to TM. Here, we present a phylogenomic analysis identifying three candidate proteins associated with membrane trafficking that may contribute to TM biogenesis, including the SPFH family member Slr1106, which we show was acquired via lateral gene transfer. Moreover, evolutionary analysis of 36 PSII assembly factors indicates key modifications in late-stage PSII assembly, notably in manganese homeostasis. We further highlight structural changes in the early-acting YidC translocase that may have facilitated the relocation of LET components from the CM to TM. Altogether, our phylogenetic and functional prediction analyses of proteins involved in membrane dynamics and PSII assembly factors bring new insights into the molecular innovations that led to the emergence of TM.

N-acetyl L-leucine (NALL, USAN or levacetylleucine, INN or trade name Aqneursa) is an FDA-approved agent for the treatment of Niemann-Pick disease type C (NPC). The N-acetyl group renders the compound a prodrug of L-leucine, making it a substrate for membrane-spanning monocarboxylate transporters (MCTs), which are ubiquitously expressed delivering NALL to all tissues with high capacity, including to the central nervous system. NALL enters enzyme-controlled pathways that correct metabolic dysfunction and enhance mitochondrial bioenergetics. Because NALL improves energy production (adenosine triphosphate, ATP) and ameliorates lysosomal function, it is potentially a therapy for a broad range of neurodegenerative and neurodevelopmental disorders (in addition to lysosomal storage disorders) in which energy homeostasis and lysosomal function are impaired. Here, we have performed a series of in vitro studies which reveal an additional aspect of NALLs polypharmacological mechanism of action. The studies demonstrate a direct lysosomal effect whereby NALL rapidly activates the translocation of the Transcription Factor EB (TFEB, a master regulator of lysosomal biogenesis and autophagy) from the cytoplasm to the nucleus in HeLa cells. The activation of TFEB is known to trigger the activation of specific genes that restore lysosomal biogenesis and function, as well as autophagy. Consistent with this, we show that NALL increases production of a TFEB target gene LAMP1, an integral lysosomal membrane protein responsible for maintaining lysosomal integrity, function and pH. This in vitro effect occurs at concentrations consistent with concentrations in plasma in humans after standard therapeutic dosing. We further demonstrated that acetylation is critical to this aspect of NALLs mechanism of action, as L-leucine itself had no effect on the activation of TFEB. Consistent with previous studies N-acetyl-D-leucine was inactive and also had no effect. Similarly, N-acetyl-DL-leucine also had only a modest effect, providing further evidence that N-acetyl-D-leucine is even antagonistic and inhibits the effects of the active L-enantiomer. This mechanism of action of NALL to activate TFEB signalling, thereby enhancing lysosomal and autophagic function, further elucidates the ways by which this compound targets the fundamental etiology of rare and common neurodegenerative disorders, from Niemann-Pick disease type C to Parkinsons disease. Based on its mechanism of action by improving the mitochondrial-lysosomal axis, NALL has the potential to be an effective therapy for a broad range of neurological and neurodevelopment conditions.

Circadian clocks regulate biological activities, providing organisms a fitness advantage under diurnal changing conditions by allowing them to anticipate and adapt to recurring external changes. In recent years attention was drawn to the entrainment by intracellular cycles. Photosynthetic Cyanobacteria coordinate their gene expression, metabolism, and other activities in a circadian fashion. Solely, three proteins, KaiA, KaiB, and KaiC, constitute the well-studied circadian clock of the cyanobacterial model, Synechococcus elongatus PCC 7942. It remained inconclusive for a long time whether Synechocystis sp. PCC 6803, an important organism for biotechnological applications, can also maintain circadian rhythms under continuous illumination. Using an approach, which does not require genetic modification, we investigated the growth behavior of Synechocystis via non-invasive online backscattering measurement and verified all three criteria for true circadian oscillators: temperature compensation, entrainment by external stimuli, and a self-sustained freerunning period of about 24 hours. Since manipulation of the circadian clock (Synechocystis {Delta}kaiA1B1C1) led to a significant reduction in glycogen content, disruption of glycogen synthesis (Synechocystis {Delta}glgC) entirely inhibited glycogen formation and both mutants lost oscillations, we hypothesize that the oscillations reflect glycogen metabolism. Significance StatementMonitoring circadian rhythms in cyanobacteria usually requires genetically modified reporter strains or intensive sampling for downstream analysis. Even for the main cyanobacterial model Synechocystis sp. PCC 6803 it was debated for years to which extent undamped circadian oscillations are really present until a suitable reporter strain was developed. We applied online backscatter measurements as an alternative readout to monitor circadian oscillations in cyanobacteria. In Synechocystis the temperature-compensated kaiA1B1C1-driven 24 h metabolic oscillations did not require light-dark entrainment, highlighting the relevance of the clock for the carbon metabolism even under continuous light, an aspect which should be considered for industrial set-ups. Our method opens the possibility to extend circadian analysis to non-GMO and monitor metabolic rhythmicity during high-density cultivation.

The diversity of marine cyanobacteria has been extensively studied due to their vital roles in ocean primary production. However, little is understood about the diversity of cyanobacterial species involved in symbiotic relationships. In this study, we successfully sequenced the complete genome of a cyanobacterium in symbiosis with Citharistes regius, a dinoflagellate species thriving in the open ocean. A phylogenomic analysis revealed that the cyanobacterium (CregCyn) belongs to the marine picocyanobacterial lineage, akin to another cyanobacterial symbiont (OmCyn) of a different dinoflagellate closely related to Citharistes. Nevertheless, these two symbionts are distinct lineages, suggesting independent origins of their symbiotic lifestyles. Despite the distinct origins, the genome analyses of CregCyn revealed shared characteristics with OmCyn, including an obligate symbiotic relationship with the host dinoflagellates and a degree of genome reduction. In contrast, a detailed analysis of genome subregions unveiled that the CregCyn genome carries genomic islands that are not found in the OmCyn genome. The presence of the genomic islands implies that exogenous genes have been integrated into the CregCyn genome at some point in its evolution. This study contributes to our understanding of the complex history of the symbiosis between dinoflagellates and cyanobacteria, as well as the genomic diversity of marine picocyanobacteria.

Group II introns (G2Is) are self-splicing ribozymes that have retroelement characteristics in prokaryotes. Although G2Is are considered an important factor in the evolution of prokaryotes, comprehensive analyses of these introns among the tens of thousands of prokaryotic genomes currently available are still limited. Here, we developed a bioinformatic pipeline that systematically collects G2Is and applied it to prokaryotic genomes. We found that in bacteria, 25% (447 of 1,790) of the total representative species had an average of 5.3 G2Is, and in archaea, 9% (28 of 296) of the total representative species had an average of 3.0 G2Is. The greatest number of G2Is per species was 101 in Arthrospira platensis (phylum Cyanobacteriota). A comprehensive sequence analysis of the intron-encoded protein (IEP) in each G2I sequence was conducted and resulted in the addition of three new IEP classes (U1-U3) to the previous classification. This analysis suggested that about 30% of all IEPs are noncanonical IEPs. The number of G2Is per species was defined almost at the phylum level, and the type of IEP was associated as a factor in the G2I increase, i.e. there was an explosive increase in G2Is with bacterial C-type IEPs in the phylum Firmicutes and in G2Is with CL-type IEPs in the phylum Cyanobacteriota. We also systematically analyzed the relationship between genomic signatures and the mechanism of these increases in G2Is. This is the first study to systematically characterize G2Is in the prokaryotic phylogenies.