Proteogenomics is a transformative approach for deciphering novel coding regions through integration of genomic, transcriptomic, and proteomic data. Here, we present pAnno, an end-to-end workflow designed to uncover hidden protein-coding elements with high precision and efficiency. pAnno generates customized protein databases by integrating multi-omic data, employs a multi-stage iterative open search strategy, and incorporates an efficient peptide-to-coding sequence mapping algorithm. Despite a 50-fold increase in database size, pAnno maintains high sensitivity and accuracy in peptide identification and achieves genomic localization of novel events with only ∼ 3

Mycobacterium tuberculosis is the causative pathogen of human tuberculosis and a leading cause of death worldwide attributed to a single infectious agent. While small non-coding RNAs (sRNAs) have emerged as key regulators of bacterial pathogenicity, their specific roles and mechanisms in M. tuberculosis remain poorly understood. Here, we employed label-free quantitative proteomics, parallel reaction monitoring, and sRNA-seq analyses to identify proteomic differences between the virulent H37Rv and attenuated H37Ra strains. Bioinformatic analysis revealed significant enrichment of differentially expressed proteins involved in lipid metabolism and fatty acid biosynthesis, key pathways linked to M. tuberculosis virulence. We identified a novel sRNA, ASpks2, which was significantly downregulated in H37Rv. Functional validation demonstrated that ASpks2 directly targets the polyketide synthase 2 (pks2), modulating its expression to enhance M. tuberculosis survival in human macrophages THP-1 cells. By correlating the omics data with functional studies, this study identified a novel sRNA and its regulatory network in M. tuberculosis, which provides novel insight into the molecular pathogenesis of M. tuberculosis and may serve as a basis for the development of targeted therapies.

Chloromonas typhlos is a cosmopolitan alpine snow alga distributed across continents, and its blooming accelerates snow melting by decreasing the amount of snow albedo. To elucidate the genetic traits underlying the adaptation of C. typhlos to the alpine habitat, we combined PacBio sequencing and Hi-C to generate a high-quality chromosome-level genome assembly (contig N50: 1.29 Mb; scaffold N50: 7.23 Mb) with 31 chromosomes and a genome size of 200.86 Mb. Repetitive elements constituted 11.05% of the genome, and 16,133 protein-coding genes were predicted, of which 82% were functionally annotated. This study provides a set of omics resources both for snow algae and the genus Chloromonas.

Viruses commonly evade host immunity by directly targeting and destroying positive regulators of interferon (IFN) signaling. Here, we reveal an indirect and stealthy immune evasion strategy employed by a fish virus, whereby the Spring Viraemia of Carp Virus (SVCV) phosphoprotein (P protein) antagonizes host IFN responses by upregulating the host negative regulator RIOK3. Proteomics analysis identified significant upregulation of the kinase RIOK3 upon SVCV infection. We demonstrate that fish RIOK3 negatively regulates type I IFN by recruiting the selective autophagy receptor p62 to mediate degradation of TBK1. Furthermore, the SVCV P protein stabilizes RIOK3 protein levels through the E3 ubiquitin ligase TRIM11. Functional assays confirmed that the inhibition of IFN by the P protein is enhanced by RIOK3 overexpression and significantly diminished upon RIOK3 knockdown. Our findings uncover a mechanism where SVCV P protein hijacks the host negative regulator RIOK3 to suppress IFN production indirectly, representing an efficient and stealthy viral immune evasion strategy.

Lysine acetylation (Kac) is a critical post-translational modification that regulates photosynthesis and carbon metabolism in cyanobacteria. However, the diversity and functional roles of lysine acetyltransferases (KATs) beyond the well-characterized cGNAT2 remain poorly defined. This study identifies and functionally characterizes the previously unannotated protein A0096, designated here as cKAT, demonstrating its capacity to catalyze Kac both in vivo and in vitro in Synechococcus sp. PCC 7002 (Synechococcus). Deletion of cKAT significantly impaired cellular growth and photosynthetic efficiency in Synechococcus. Utilizing label-free quantitative acetylome profiling, we identified 171 endogenous Kac sites across 137 proteins targeted by cKAT. These target proteins participate in diverse metabolic and photosynthetic pathways, indicating a broad regulatory role for cKAT in cellular physiology. Notably, ChpX, a key component of CO2-concentrating mechanisms, was prominently acetylated by cKAT both in vivo and in vitro. We further established that cKAT specifically mediates acetylation at residue K88 of ChpX, a modification that directly modulates CO2 uptake efficiency. This regulatory mechanism consequently influences photosynthetic performance and cellular growth in Synechococcus. Collectively, these findings establish cKAT as a central regulator of cyanobacterial carbon fixation. This work expands the known repertoire of photosynthetic acetyltransferases and provides mechanistic insights into the Kac-dependent regulation of photosynthetic processes.

Synechocystis sp. PCC 6803 (Synechocystis), a unicellular and motile cyanobacterium, exhibits directed motility toward or away from light stimuli through type IV pili (T4P)-mediated locomotion. However, the regulatory mechanisms governing T4P-dependent motility in Synechocystis remain incompletely understood. In this study, we demonstrated that Sll0171 can catalyze lysine methylation both in vivo and in vitro. Notably, the loss of sll0171 resulted in enhanced motility, accompanied by impaired photosynthetic capacity. We identified 65 endogenous methylation sites targeted by Sll0171 in Synechocystis via label-free quantitative proteomic analysis. Subsequent functional characterization revealed that Sll0171 specifically methylates lysine 168 (K168) of PilA1, the primary structural subunit of T4P. To elucidate the role of PilA1 methylation, we employed site-directed mutagenesis to generate mutants mimicking constitutively methylated (pilA1K168Q) or nonmethylated (pilA1K168R) states. Compared to wild-type Synechocystis, the pilA1K168R mutant displayed markedly more T4P and enhanced motility, whereas the pilA1K168Q mutant exhibited near-complete inhibition of T4P and motility. Collectively, these findings identify Sll0171 as a previously uncharacterized lysine methyltransferase in Synechocystis and reveal a methylation-dependent regulatory mechanism governing T4P and motility in cyanobacteria. This work advances our understanding of post-translational modifications in modulating microbial motility and environmental adaptation.

RNA modifications have been found in all domains of life and play regulatory roles in diverse biological processes. However, their distribution, function, and regulation in cyanobacteria remain unexplored. Here, we have employed a quantitative RNA profiling strategy based on mass spectrometry analysis to identify 21 different RNA modifications in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Mass spectrometry analyses reveal a dynamic pattern of these RNA modifications under different culture conditions. We subsequently perform transcriptome-wide 5-methylcytosine (m5C) profiling in Synechocystis by using bisulfite sequencing. In total, we identify 824 high-confidence m5C sites in 382 mRNAs, with the majority of m5C-modified genes participating in ribosome, RNA degradation, carbon metabolism, and photosynthesis. Combined with the m5C-RNA immunoprecipitation detection method, 40.17% (331) m5C sites were validated and located within 129 m5C-RNA immunoprecipitation peaks on 145 mRNAs. Notably, integrated transcriptomic, proteomic, and m5C methylome analysis shows that m5C modification is negatively associated with protein abundance and contributes to the RNA-protein discordance, implying the importance of m5C on post-transcriptional regulation in Synechocystis. Collectively, our study provides a holistic view of RNA modifications and the first mRNA m5C map in cyanobacteria, which present a critical database for functional analyses of RNA modifications in cyanobacteria. The method used in this study is applicable to any sequenced prokaryotes and could be applied as a standard part of transcriptomic analysis.

Astaxanthin, a potent antioxidant carotenoid, is primarily synthesized by the microalga Haematococcus pluvialis (H. pluvialis) and has widespread applications in the nutraceutical, pharmaceutical, and aquaculture industries. In this study, we employed a tandem mass tag (TMT)-based quantitative proteomics approach to investigate the metabolic and regulatory mechanisms underlying astaxanthin biosynthesis in H. pluvialis under weak light stress conditions. Microscopic observation revealed that H. pluvialis retained its green motile morphology while progressively accumulating astaxanthin (0.22 % of dry weight by 48 h) under weak light stress conditions, without compromising growth Proteomic analysis identified 3, 57, and 311 differentially expressed proteins (DEPs) at 12, 24, and 48 h, respectively, highlighting dynamic shifts in lipid metabolism and energy production, and notably the upregulation of proteins involved in both lipid and carotenoid biosynthesis. Three DEPs-farnesyl pyrophosphotransferase (FDFT1), isocitrate dehydrogenase (IDH), and dihydropyrimidine dehydrogenase (DPD)- were selected to assess their contribution to astaxanthin biosynthesis based on expression trends and biological relevance. Due to the difficulty of genetic manipulation in H. pluvialis, heterologous expression was conducted in an engineered Escherichia coli (BW-ASTA) strain producing free astaxanthin. FDFT1 and IDH enhanced astaxanthin yields by 5.0-fold and 1.6-fold, respectively, while DPD showed no significant effect. These findings reveal proteome-level adaptations in H. pluvialis under weak light stress conditions and identify candidate genes for metabolic engineering of astaxanthin biosynthesis, laying groundwork for the development of sustainable, industrial-scale production strategies production.

Microalgae are a rich source of high-value natural products. The green microalga Chlamydomonas reinhardtii has long been used as a model organism for studying lipid metabolism in photosynthetic organisms. Here, we comprehensively characterized the enzymatic activity and substrate preferences of the plastidial glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT1) from C. reinhardtii. Our results revealed that, in addition to GPAT activity, recombinant GPAT1 is associated with lysophosphatidic acid: acyl-CoA acyltransferase (LPAAT) activity. Notably, the membrane-bound form of GPAT1 displayed distinct acyl-donor preferences, favoring both C18:1 and C16:0 substrates in its LPAAT function. Knockdown of GPAT1 resulted in a reduced triacylglycerol content, particularly C16 species, under mixotrophic growth and nitrogen deprivation. Interestingly, GPAT1 knockdown triggered a compensatory upregulation of the endoplasmic reticulum-localized GPAT2, resulting in a significant increase in the content and yield of 1,3-olein-2-palmitin (OPO), an essential functional lipid used in infant formula. These findings provide insights into the function and physiological role of microalgal plastidial GPAT1 and highlight its potential as a biotechnological target for enhancing OPO production in microalgae.

Lysine crotonylation (Kcr) is a newly identified posttranslational modification that plays an important role in diverse biological processes; however, its distribution, function, and regulation in photosynthetic organisms remain largely unknown. Cyanobacteria are the most ancient prokaryotes capable of oxygenic photosynthesis and play a vital role in global carbon and nitrogen cycles. We examined all predicted Kcr regulatory enzymes in the model cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) using total protein Kcr modification levels and enzymatic activity assays. We then used a label-free quantitative (LFQ) proteomic approach following enrichment for crotonylated peptides to identify the endogenous substrates of these Kcr regulatory enzymes. We found that cGNAT2 functions as a lysine crotonyltransferase, whereas CddA acts as a decrotonylase. Using LFQ crotonylome analysis, we identified a total of 536 endogenous Kcr sites catalyzed by cGNAT2 and 360 candidate sites targeted by CddA, with the associated proteins predominantly involved in metabolic processes and photosynthesis. Furthermore, we validated that cGNAT2 and CddA regulate the Kcr level of the Photosystem I subunit II (PsaD). cGNAT2 and CddA may influence the structure of PsaD by modulating its Kcr status or by cumulative modification effects, thereby affecting cell growth and the efficiency of photosynthetic electron transport.

Cyanobacteria are ancient and abundant photosynthetic prokaryotes that play crucial roles in global carbon and nitrogen cycles. They exist in a variety of environments and have been used extensively as model organisms for studies of photosynthesis and environmental adaptation. Lysine acetylation (Kac), a widespread and evolutionarily conserved protein posttranslational modification, is reversibly catalyzed by lysine acetyltransferases (KAT) and lysine deacetylases (KDACs). Over the past decade, a growing number of acetylated proteins have been identified in cyanobacteria, and Kac is increasingly recognized as having essential roles in many cellular processes, such as photosynthesis, energy metabolism, and stress responses. Recently, cGNAT2 and CddA were identified as KAT and KDAC in the model cyanobacterium Synechococcus sp. PCC 7002, respectively. The identified Kac regulatory enzymes provide novel insight into the mechanisms that globally regulate photosynthesis in cyanobacteria and potentially other photosynthetic organisms. This review summarizes recent progress in our understanding of the functions and mechanisms of lysine acetylation in Cyanobacteria. The challenges and future perspectives in this field are also discussed.

Tetrahymena thermophila (T. thermophila), a well-established model organism, has been instrumental in advancing our understanding of evolutionarily conserved biological processes. A key biological feature of this unicellular eukaryote is its life cycle strategy, marked by three major stages: growth, starvation, and conjugation. Despite its prominence as a model system, functional genomic studies of T. thermophila have been constrained by limitations in the accuracy and completeness of gene discovery since the initial genome assembly in 2006. To address this gap, we performed a multi-stage proteogenomic analysis, combining genomic sequencing with high-resolution mass spectrometry (MS)-based proteomic profiling across 10 strategically selected life cycle states. This integrative approach enabled a comprehensive reassessment of gene discovery, leading to the validation of 24,319 previously predicted protein-coding genes and the identification of 383 novel genes. Additionally, our investigation systematically identified a diverse repertoire of post-translational modifications (PTMs), including 7123 modification sites distributed across 4705 proteins. These PTMs are postulated to exert critical regulatory functions during developmental phase transitions. Collectively, this work not only refines the T. thermophila gene catalog and enhances its utility as a robust genetic toolkit for advancing biological research but also offers new mechanistic insights into the molecular regulation of its life cycle progression.

Ciliates represent a diverse assemblage of ancient single-celled eukaryotes characterized by diverse morphological features. Among certain sessilid peritrich ciliates, an exceptional morphological structure known as the stalk has been documented since the pioneering work of Antonie van Leeuwenhoek in the 17th century. This study conducts a comparative genomic analysis of three sessile peritrich species-Epistylis sp., Vorticella campanula, and Zoothamnium arbuscula-and two free-swimming species, Tetrahymena thermophila and Paramecium tetraurelia, within the class Oligohymenophorea. We find that carbohydrate-related components are consistently associated with diverse stalk substructures. Evidence suggests that the branched stalks of colonial E. hentscheli are supported by chitin-based ring-like structures. Through proteomic analysis of the Epistylis stalk, we found peritrich-specific genes, including coiled-coil domain-containing (CCDC) proteins and epidermal growth factor-like (EGF-like) proteins, as key stalk components. CCDC proteins are part of the stalk sheath, and their N-glycosylation may enhance adhesion between the cell body and stalk through lectin interactions. This study sheds light on the genetic innovations behind the stalk in peritrichs, which support their sessile and colonial lifestyles, and identifies peritrich-specific CCDC proteins as potential targets for disrupting the attachment of sessilids to aquaculture animals, addressing issues related to epibiotic burden.

Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "neck" that can stretch 7-8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space search and prey capture. This remarkable morphological change involves a unique actin-myosin system rather than the Ca2+-dependent system found in other contractile ciliates. Two cytoskeletons are identified in the cortex of the Lacrymaria cell, namely the myoneme cytoskeleton and the microtubule cytoskeleton. The myoneme cytoskeleton is composed of centrin-myosin proteins, exhibiting distinct patterns between the neck and body, with their boundary seemingly associated with the position of the macronucleus. A novel giant protein forming a ladder-like structure was discovered as a component of the microtubule cytoskeleton. Thick centrin-myosin fibers are situated very close to the right side of the ladders in the neck but are far away from such structures in the body. This arrangement enables the decoupling of the neck and body. Plasmodium-like unconventional actin has been discovered in Lacrymaria, and this may form highly dynamic short filaments that could attach to the giant protein and myosin, facilitating coordination between the two cytoskeletons in the neck. In summary, this fascinating organism employs unconventional cytoskeletal components to accomplish its extraordinary dynamic shape-shifting.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H2O2 resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

Lysine acetylation is a conserved regulatory posttranslational protein modification that is performed by lysine acetyltransferases (KATs). By catalyzing the transfer of acetyl groups to substrate proteins, KATs play critical regulatory roles in all domains of life; however, no KATs have yet been identified in cyanobacteria. Here, we tested all predicted KATs in the cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) and demonstrated that A1596, which we named cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2), can catalyze lysine acetylation in vivo and in vitro. Eight amino acid residues were identified as the key residues in the putative active site of cGNAT2, as indicated by structural simulation and site-directed mutagenesis. The loss of cGNAT2 altered both growth and photosynthetic electron transport in Syn7002. In addition, quantitative analysis of the lysine acetylome identified 548 endogenous substrates of cGNAT2 in Syn7002. We further demonstrated that cGNAT2 can acetylate NAD(P)H dehydrogenase J (NdhJ) in vivo and in vitro, with the inability to acetylate K89 residues, thus decreasing NdhJ activity and affecting both growth and electron transport in Syn7002. In summary, this study identified a KAT in cyanobacteria and revealed that cGNAT2 regulates growth and photosynthesis in Syn7002 through an acetylation-mediated mechanism.

The giant single-celled eukaryote, Spirostomum , exhibits one of the fastest movements in the biological world. This ultrafast contraction is dependent on Ca 2+ rather than ATP and therefore differs to the actin-myosin system in muscle. We obtained the high-quality genome of Spirostomum minus from which we identified the key molecular components of its contractile apparatus, including two major Ca 2+ binding proteins (Spasmin 1 and 2) and two giant proteins (GSBP1 and GSBP2), which act as the backbone and allow for the binding of hundreds of spasmins. The evidence suggests that the GSBP-spasmin protein complex is the functional unit of the mesh-like contractile fibrillar system, which, coupled with various other subcellular structures, provides the mechanism for repetitive ultrafast cell contraction and extension. These findings improve our understanding of the Ca 2+ -dependent ultrafast movement and provide a blueprint for future biomimicry, design, and construction of this kind of micromachine.

Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "cell neck" that can stretch 7-8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space-search and prey-capture. This remarkable morphological change involves a unique actin-myosin system rather than the Ca2+-dependent system found in other contractile ciliates. Two cytoskeletons have been identified in the cortex of the Lacrymaria cell, namely the myoneme cytoskeleton and the microtubule cytoskeleton. The myoneme cytoskeleton is composed of centrin-myosin proteins that display distinct patterns between the neck and body, with their boundary seemingly determined by the macronucleus. A novel giant protein forming a ladder-like structure was discovered as a component of the microtubule cytoskeleton. Thick centrin-myosin fibers are situated very close to the right side of the ladders in the neck but are far away from such structures in the body. This arrangement enables the decoupling of the neck and body. Plasmodium-like unconventional actin has been discovered in Lacrymaria, and this may form highly dynamic short filaments that could attach to the giant protein and myosin, facilitating coordination between the two cytoskeletons in the neck. In summary, this fascinating organism employs unconventional and novel cytoskeletal components to accomplish its extraordinary dynamic shape-shifting.

[Background] Proteases can degrade the misfolded or nonfunctional proteins in cells. Clp family protein is one of the important protease complexes. ClpP is the core of proteolysis of Clp protease complex. According to genomic data, there are four different ClpP proteins in Synechocystis sp. PCC6803, namely ClpP1–ClpP4. As a vital functional component of proteolytic complex, ClpP in Synechocystis is currently poorly studied. The study on its physiological function and substrate regulation is limited. [Objective] To explore the functions of ClpP2 in Synechocystis and identify potential substrate clusters, thereby providing experimental support for the mechanism research of ClpP2. [Methods] The ClpP2 mutant strain（ΔClpP2） was constructed, and the growth experiment and photosynthetic system characteristic experiment were carried out. Target proteins regulated by ΔClpP2 were identified by isobaric tag for relative absolute quantitation（iTRAQ）, and the metabolic pathways involved in the substrate proteins were analyzed via bioinformatics. Finally, parallel reaction monitoring（PRM）was used to verify part of the quantitative data. [Results] ΔClpP2 grew into the logarithmic phase through photoautotrophy under natural conditions, but did not grow normally when met high-light or high-temperature stress. Compared with the wild-type（WT）, ΔClpP2 showed significantly reduced photosystem Ⅱ（PSⅡ） electron transport efficiency and circle electron transport activity of photosystem Ⅰ（PSⅠ）. A total of 206 differentially expressed proteins in ΔClpP2 were identified by iTRAQ quantitative proteomics. Among them, 131 were up-regulated and 74 were down-regulated, which provided a rich substrate library. Gene Ontology（GO） analysis showed that ClpP2 was mainly involved in the transport of various substances, and ABC transporter pathway was enriched notably. Thirty-four differentially expressed proteins were successfully verified by PRM technology. [Conclusion] ClpP2 is not necessary for the growth of Synechocystis, but is essential when Synechocystis meets high-temperature or high-light stress. ClpP2 inactivation reduces the activity of the photosynthetic system in Synechocystis. ClpP2 might affect the photosynthetic system by regulating ion transport. ClpP2 is likely to bind with ClpX to form a protease complex.