Large Subunit Protein Research Articles

Critical stages in pea photosynthesis impaired by tetracycline as an environmental contaminant.

The widespread use of antibiotics in intensive animal husbandry, and the agricultural utilization of manure from such farms, imposes a significant burden on the environment. Consequently, the effects of antibiotics should be studied not only in animals and humans but also in all components of biocenoses and agrocenoses. In our study, we analyze the impact of four different concentrations of tetracycline present in soil (0, 5, 50, and 500mg/kg of soil) on the growth and key photosynthesis parameters of pea seedlings: chlorophyll concentration, aminolevulinic acid concentration, aminolevulinic acid dehydrogenase activity, and ribulose bisphosphate carboxylase-oxygenase (RuBisCO) activity. At the lowest tetracycline concentration, chlorophyll content decreased by 13% compared to the control (0 tetracycline), while at the highest antibiotic concentration, it decreased by as much as 27%. Similarly, the decrease in aminolevulinic acid (a chlorophyll precursor) concentration was significant, amounting to 34%. However, the activity of the dehydrogenase enzyme, which consumes this precursor, decreased even more drastically by 51%, indicating significant disturbances in the light phase of photosynthesis. However, the activity of RuBisCO in pea plants subjected to tetracycline was even more severely affected, dropping by 58%, 69%, and 70% in soils with increasing concentrations of tetracycline. The reduction in enzyme activity could only partially be explained by a less pronounced decrease in the quantity of RuBisCO (large subunit) protein, which amounted to 6.5%, 11%, and 35% for tetracycline concentrations of 5, 50, and 500mg/kg of soil, respectively.

Abstract 3100: BRCA1-RpL17 Interaction Regulates Angiogenesis

Background: Angiogenesis, forming new blood vessels from existing ones, is crucial in development, diabetes, and cancer. We found that Large Ribosomal subunit protein 17 (RpL17) played a key role in endothelial cell (EC) proliferation and angiogenesis. Reduced RpL17 (KD) promoted angiogenesis in human ECs and RpL17-deficient mouse retinas. RNA-sequencing and bioinformatics suggested a role for Early growth response 1(EGR1) transcription factor. Bioinformatics revealed Breast Cancer gene 1 (BRCA1) as a potential upstream regulator of RpL17, suggesting a novel pathway for angiogenesis. Hypothesis: BRCA1 interacts with RpL17 and upregulates EGR1 to stimulate vessel elongation in angiogenesis. Goal: Determine the role of BRCA1-RpL17 interaction in angiogenesis and their potential feedback loop via EGR1. Methods/Approach: RNA-sequencing of mouse ECs, whole retina mount staining (comparing wild-type and RpL17+/- mice). Tube formation assays with HUVECs to investigate angiogenesis after RpL17, BRCA1, and EGR1 knockdowns. Bioinformatics analysis with Python for RNA-seq data. Immunoblotting and RT-qPCR to measure protein and DNA expression. Immunofluorescence (IF) staining of HUVECs to observe BRCA1, RpL17, and EGR1 expression and localization. Preliminary Results: Whole retina mounts showed increased angiogenesis in RpL17-deficient mouse retinas compared to wildtype (p<0.009). HUVECs with RpL17 KD had increased junctions and longer branches than control HUVECs (p<0.0002). RNA-seq identified increased EGR1 expression (q<2.5x10 -8 ) in RpL17 KD ECs, confirmed with RT-qPCR (p<0.002). Bioinformatics predicted BRCA1 as a regulator of RpL17. HUVEC IF staining showed decreased RpL17 expression after BRCA1 KD. Angiogenesis assays of BRCA1 KD ECs exhibited an angiogenic phenotype like RpL17 KD, rescued following double BRCA1/RpL17 KD. Conclusion: BRCA1-RpL17 interaction stimulates angiogenesis and may be regulated by an EGR1 feedback loop, likely by EC proliferation. Detailed understanding of this pathway will provide new therapies to regulate angiogenesis.

Dynamic protein composition of Saccharomyces cerevisiae ribosomes in response to multiple stress conditions reflects alterations in translation activity

Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.

The zinc finger motif in the mitochondrial large ribosomal subunit protein bL36m is essential for optimal yeast mitoribosome assembly and function

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465.

Extracellular Proteins Isolated from L. acidophilus as an Osteomicrobiological Therapeutic Agent to Reduce Pathogenic Biofilm Formation, Regulate Chronic Inflammation, and Augment Bone Formation In Vitro.

Periprosthetic joint infection (PJI) is a challenging complication that can occur following joint replacement surgery. Efficacious strategies to prevent and treat PJI and its recurrence remain elusive. Commensal bacteria within the gut convey beneficial effects through a defense strategy named "colonization resistance" thereby preventing pathogenic infection along the intestinal surface. This blueprint may be applicable to PJI. The aim is to investigate Lactobacillus acidophilus spp. and their isolated extracellular-derived proteins (LaEPs) on PJI-relevant Staphylococcus aureus, methicillin-resistant S. aureus, and Escherichia coli planktonic growth and biofilm formation in vitro. The effect of LaEPs on cultured macrophages and osteogenic, and adipogenic human bone marrow-derived mesenchymal stem cell differentiation is analyzed. Data show electrostatically-induced probiotic-pathogen species co-aggregation and pathogenic growth inhibition together with LaEP-induced biofilm prevention. LaEPs primemacrophages for enhanced microbial phagocytosis via cathepsin K, reduce lipopolysaccharide-induced DNA damage and receptor activator nuclear factor-kappa B ligand expression, and promote a reparative M2 macrophage morphology under chronic inflammatory conditions. LaEPs also significantly augment bone deposition while abating adipogenesis thus holding promise as a potential multimodal therapeutic strategy. Proteomic analyses highlight high abundance of lysyl endopeptidase, and urocanate reductase. Further, in vivo analyses are warranted to elucidate their role in the prevention and treatment of PJIs.

Chloroplast-targeted late embryogenesis abundant 1 increases alfalfa tolerance to drought and aluminum.

Late embryogenesis-abundant (LEA) proteins are important stress-response proteins that participate in protecting plants against abiotic stresses. Here, we investigated LEA group 3 protein MsLEA1, containing the typically disordered and α-helix structure, via overexpression and RNA interference (RNAi) approaches in alfalfa (Medicago sativa L.) under drought and aluminum (Al) stresses. MsLEA1 was highly expressed in leaves and localized in chloroplasts. Overexpressing MsLEA1 increased alfalfa tolerance to drought and Al stresses, but downregulating MsLEA1 decreased the tolerance. We observed a larger stomatal aperture and a lower water use efficiency in MsLEA1 RNAi lines compared with wild-type plants under drought stress. Photosynthetic rate, Rubisco activity, and superoxide dismutase (SOD) activity increased or decreased in MsLEA1-OE or MsLEA1-RNAi lines, respectively, under drought and Al stress. Copper/zinc SOD (Cu/Zn-SOD), iron SOD (Fe-SOD), and Rubisco large subunit proteins (Ms1770) were identified as binding partners of MsLEA1, which protected chloroplast structure and function under drought and Al stress. These results indicate that MsLEA1 recruits and protects its target proteins (SOD and Ms1770) and increases alfalfa tolerance against drought and Al stresses.

Experimental upregulation of developmentally downregulated ribosomal protein large subunits 7 and 7A promotes axon regeneration after injury in vivo

Ribosomal proteins are involved in neurodevelopment and central nervous system (CNS) disease and injury. However, the roles of specific ribosomal protein subunits in developmental axon growth, and their potential as therapeutic targets for treating CNS injuries, are still poorly understood. Here, we show that ribosomal protein large (Rpl) and small (Rps) subunit genes are substantially (56-fold) enriched amongst the genes, which are downregulated during maturation of retinal ganglion cell (RGC) CNS projection neurons. We also show that Rpl and Rps subunits are highly co-regulated in RGCs, and partially re-upregulated after optic nerve crush (ONC). Because developmental downregulation of ribosomal proteins coincides with developmental decline in neuronal intrinsic axon growth capacity, we hypothesized that Rpl/Rps incomplete re-upregulation after injury may be a part of the cellular response which attempts to reactivate intrinsic axon growth mechanisms. We found that experimentally upregulating Rpl7 and Rpl7A promoted axon regeneration after ONC in vivo. Finally, we characterized gene networks associated with Rpl/Rps, and showed that Rpl7 and Rpl7A belong to the cluster of genes, which are shared between translational and neurodevelopmental biological processes (based on gene-ontology) that are co-downregulated in maturing RGCs during the decline in intrinsic axon growth capacity.

Corrected and Republished from: "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. PA-SR01 is a member of Siphoviridae with a long noncontractile tail. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles. IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Spontaneously Produced Lysogenic Phages Are an Important Component of the Soybean Bradyrhizobium Mobilome.

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.

Unsupervised voxel-based segmentation reveals landscape of bacterial ribosome large subunit early assembly.

Ribosome biogenesis is a complex but efficient process in rapidly growing bacteria, where assemble a functional 70S ribosome takes ∼2 min, involving participation of 3 rRNAs, 50 r-proteins and dozens of assembly factors. In vitro reconstitution using different subsets of large subunit (50S, LSU) proteins with rRNAs, pioneered by Nierhaus lab, resulted in the Nierhaus assembly map, embodying the cooperativity and dependency for binding of LSU r-proteins to 23S rRNA. Critically absent from the Nierhaus map is the underlying folding of the rRNA that creates the binding sites for the r-proteins. In addition, the relationship of the observed cooperativity in vitro to the co-transcriptional assembly in cells remains to be determined. Pre-50S intermediates accumulate at low temperature in ∆deaD, a DEAD-box helicase implicated in 50S assembly. We solved 21 pre-50S density maps from intermediate-containing fractions using cryo-EM. In the newly solved maps, we discovered the earliest intermediate ever reported, consisting of domain I at the 5’-end of 23S rRNA. To probe the mechanism behind the maps during assembly, we developed a novel density map segmentation and dependency analysis method. Ten cooperative assembly blocks were identified from segmentation of the maps, and these were organized into a block dependency map. This is the first time the dependencies on folding of rRNA helices and ribosomal protein binding could be integrated into a complete assembly map. In addition, we showed how the exit tunnel is folded on the solvent side, serving as a scaffold for 50S maturation. Using this new segmentation analysis method, we revisited previously reported bL17-depletion and ∆srmB datasets. Most remarkably, the other two datasets are also consistent with the block dependency, implying a unified early assembly pathway and flexible maturation landscape in early 50S biogenesis.

DPC29 promotes post-initiation mitochondrial translation in Saccharomyces cerevisiae.

Mitochondrial ribosomes synthesize essential components of the oxidative phosphorylation (OXPHOS) system in a tightly regulated process. In the yeast Saccharomyces cerevisiae, mitochondrial mRNAs require specific translational activators, which orchestrate protein synthesis by recognition of their target gene's 5'-untranslated region (UTR). Most of these yeast genes lack orthologues in mammals, and only one such gene-specific translational activator has been proposed in humans-TACO1. The mechanism by which TACO1 acts is unclear because mammalian mitochondrial mRNAs do not have significant 5'-UTRs, and therefore must promote translation by alternative mechanisms. In this study, we examined the role of the TACO1 orthologue in yeast. We found this 29 kDa protein to be a general mitochondrial translation factor, Dpc29, rather than a COX1-specific translational activator. Its activity was necessary for the optimal expression of OXPHOS mtDNA reporters, and mutations within the mitoribosomal large subunit protein gene MRP7 produced a global reduction of mitochondrial translation in dpc29Δ cells, indicative of a general mitochondrial translation factor. Northern-based mitoribosome profiling of dpc29Δ cells showed higher footprint frequencies at the 3' ends of mRNAs, suggesting a role in translation post-initiation. Additionally, human TACO1 expressed at native levels rescued defects in dpc29Δ yeast strains, suggesting that the two proteins perform highly conserved functions.

Involvement of Target of Rapamycin (TOR) Signaling in the Regulation of Crosstalk between Ribosomal Protein Small Subunit 6 Kinase-1 (RPS6K-1) and Ribosomal Proteins

The target of rapamycin (TOR) protein phosphorylates its downstream effector p70kDa ribosomal protein S6 kinases (S6K1) for ribosome biogenesis and translation initiation in eukaryotes. However, the molecular mechanism of TOR-S6K1-ribosomal protein (RP) signaling is not well understood in plants. In the present study, we report the transcriptional upregulation of ribosomal protein large and small subunit (RPL and RPS) genes in the previously established TOR overexpressing transgenic lines of rice (in Oryza sativa ssp. indica, variety BPT-5204, TR-2.24 and TR-15.1) and of Arabidopsis thaliana (in Col 0 ecotype, ATR-1.4.27 and ATR-3.7.32). The mRNA levels of RP genes from this study were compared with those previously available in transcriptomic datasets on the expression of RPs in relation to TOR inhibitor and in the TOR-RNAi lines of Arabidopsis thaliana. We further analyzed TOR activity, i.e., S6K1 phosphorylation in SALK lines of Arabidopsis with mutation in rpl6, rpl18, rpl23, rpl24 and rps28C, where the rpl18 mutant showed inactivation of S6K1 phosphorylation. We also predicted similar putative Ser/Thr phosphorylation sites for ribosomal S6 kinases (RSKs) in the RPs of Oryza sativa ssp. indica and Arabidopsis thaliana. The findings of this study indicate that the TOR pathway is possibly interlinked in a cyclic manner via the phosphorylation of S6K1 as a modulatory step for the regulation of RP function to switch 'on'/'off' the translational regulation for balanced plant growth.

Salt-Stressed Coriander (Coriandrum sativum L.) Responses to Potassium Silicate, Humic Acid and Gamma Irradiation Pretreatments

The application of biostimulants has great potential in preserving plants against abiotic or biotic stresses and is integrated into promoting tolerance and acclimating of coriander against salinity stress. Deciphering the morphological, physiological and molecular responses underpinning the ameliorative aspects of ecofriendly biostimulants is indispensable to link and overlap the ameliorative responses of seed priming. Hereby, the ameliorative responses of seed priming against salinity stress using potassium silicate, humic acid, and gamma irradiation were evaluated. Salinity stress generally diminishes vegetation, productivity, and metabolic activities. However, abscisic acid (ABA) levels and soluble sugars were elevated. Pretreatments with potassium silicate or humic acid, followed by gamma rays, alleviated and promoted growth parameters, yield components, and vital metabolic processes in salinity-stressed coriander. This promotion was concurrent with an increase in growth promotors, chlorophyll a/b, carbohydrates, antioxidants (compounds and enzymes), and upregulation of RuBisCO large subunit protein expression. Collectively, potassium silicate and humic acid were the best at alleviating the adverse effects of saline conditions. Triggered pretreatments might be engaged in maintaining metabolic activities toward deleterious salinity impacts. Thus, it was suggested that seed priming by potassium silicate and humic acid is an effective regime benefitting salinized along with nonsalinized plants that sustain coriander productivity.

First Isolation and Characteristics of Bovine Parainfluenza Virus Type 3 from Yaks.

The yaks belong to the genus Bos within the family Bovidae that live in the Tibet Plateau and is an indispensable economic resource for the local herders. Respiratory tract infections are common diseases in yaks caused by various pathogens; however, there have been no reports of bovine parainfluenza virus type 3 (BPIV3) infection. This study was conducted to investigate the pathogens and analyze their characteristics from the four yak lung samples with severe respiratory tract infection symptoms in the yak farm. Results showed that out of four lung samples, three were identified as BPIV3-positive by RT-PCR. A BPIV3 strain (106.5 TCID50/mL) was successfully isolated from the BPIV3-positive lung samples using Madin–Darby bovine kidney cells. The isolate caused systemic infection in the BALB/c mice and induced pathological changes in the lungs. Moreover, three complete BPIV3 genomes were amplified from the clinical samples. Phylogenetic trees based on the complete genomes, hemagglutinin-neuraminidase protein (HN), phosphoprotein (P), and large polymerase subunit protein (L) amino acid sequences showed that the complete BPIV3 genomes belonged to BPIV3 genotype C, and clustered into a large branch with the Chinese strains, although the three yak BPIV3 strains were clustered into a small branch. Compared to known BPIV3 genotype C strains in GenBank, the three genomes of yak BPIV3 showed four identical amino acid mutations in the HN, P and L proteins, suggesting a unique genetic evolution of BPIV3 in yaks. This study first isolated and characterized the BPIV3 from yaks, which contributed to the understanding of the infection and evolution of BPIV3 in yaks in the Tibet Plateau.

Elp3‐mediated codon‐dependent translation promotes mTORC2 activation and regulates macrophage polarization

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1‐activating signals in myeloid cells, where it limits the production of pro‐inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2‐activating signals upregulate Elp3 expression through a PI3K‐ and STAT6‐dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon‐dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt‐driven tumor initiation in the intestine by maintaining a pool of tumor‐associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

Characterization and genomic analysis of the novel Aeromonas veronii phage pAEv1812.

Bacteria of the species Aeromonas veronii are generally ubiquitous in different aquatic environments and are capable of causing a variety of diseases in aquatic animals. In this work, the phage isolate pAEv1812, which is lytic to A. veronii strain 1810, was characterized and sequenced. Morphological analysis revealed that pAEv1812 has a symmetrical head and a long non-contractile tail. A one-step growth curve analysis showed that the latent period and burst period of pAEv1812 were approximately 40 min and 80 min, respectively, with a burst size of 190 PFU/infected cell. The genome of phage pAEv1812 is 61,461 bp in length with a G+C content of 61.4%. Eighteen of the 75 putative proteins encoded by this phage have known functions, and there were no rRNA and tRNA genes in the genome. Phylogenetic analysis based on amino acid sequences of the major capsid protein and terminase large subunit suggested that phage pAEv1812 should be included as a member of the genus Chivirus.

DeephageTP: a convolutional neural network framework for identifying phage-specific proteins from metagenomic sequencing data.

Bacteriophages (phages) are the most abundant and diverse biological entity on Earth. Due to the lack of universal gene markers and database representatives, there about 50–90% of genes of phages are unable to assign functions. This makes it a challenge to identify phage genomes and annotate functions of phage genes efficiently by homology search on a large scale, especially for newly phages. Portal (portal protein), TerL (large terminase subunit protein), and TerS (small terminase subunit protein) are three specific proteins of Caudovirales phage. Here, we developed a CNN (convolutional neural network)-based framework, DeephageTP, to identify the three specific proteins from metagenomic data. The framework takes one-hot encoding data of original protein sequences as the input and automatically extracts predictive features in the process of modeling. To overcome the false positive problem, a cutoff-loss-value strategy is introduced based on the distributions of the loss values of protein sequences within the same category. The proposed model with a set of cutoff-loss-values demonstrates high performance in terms of Precision in identifying TerL and Portal sequences (94% and 90%, respectively) from the mimic metagenomic dataset. Finally, we tested the efficacy of the framework using three real metagenomic datasets, and the results shown that compared to the conventional alignment-based methods, our proposed framework had a particular advantage in identifying the novel phage-specific protein sequences of portal and TerL with remote homology to their counterparts in the training datasets. In summary, our study for the first time develops a CNN-based framework for identifying the phage-specific protein sequences with high complexity and low conservation, and this framework will help us find novel phages in metagenomic sequencing data. The DeephageTP is available at https://github.com/chuym726/DeephageTP.

History-dependent physiological adaptation to lethal genetic modification under antibiotic exposure.

Genetic modifications, such as gene deletion and mutations, could lead to significant changes in physiological states or even cell death. Bacterial cells can adapt to diverse external stresses, such as antibiotic exposure, but can they also adapt to detrimental genetic modification? To address this issue, we visualized the response of individual Escherichia coli cells to deletion of the antibiotic resistance gene under chloramphenicol (Cp) exposure, combining the light-inducible genetic recombination and microfluidic long-term single-cell tracking. We found that a significant fraction (∼40%) of resistance-gene-deleted cells demonstrated a gradual restoration of growth and stably proliferated under continuous Cp exposure without the resistance gene. Such physiological adaptation to genetic modification was not observed when the deletion was introduced in 10 hr or more advance before Cp exposure. Resistance gene deletion under Cp exposure disrupted the stoichiometric balance of ribosomal large and small subunit proteins (RplS and RpsB). However, the balance was gradually recovered in the cell lineages with restored growth. These results demonstrate that bacterial cells can adapt even to lethal genetic modifications by plastically gaining physiological resistance. However, the access to the resistance states is limited by the environmental histories and the timings of genetic modification.

Abstract 106: Ribosomal Protein Rpl17 Controls Endothelial Cell Translation: A Key Mediator Of Intima Growth And Vascular Disease

Background: Carotid intima-media thickening (IMT) is a significant risk factor for cardiovascular disease (CVD). Using genetic analysis of the partial carotid ligation (PCL) model of disturbed flow induced IMT, we identified a strong association between decreased expression of the large ribosomal subunit protein 17 (RpL17) and increased IMT. RpL17 is located at the exit tunnel of the ribosome, making it critical for processing of newly translated proteins. To define the mechanism by which RpL17 inhibits IMT, we generated EC-restricted (Cdh5-Cre) conditional RpL17 heterozygous mice (EC-RpL17). Results: 1) PCL of EC-RpL17 increased IMT vs wild type (Control) littermates. 2) EM analysis revealed decreases in total ribosome number and polysomes in EC-RpL17, consistent with decreased global translation. 3) RNA-seq showed major changes in two pathways: Eukaryotic Initiation Factor 2 (eIF2α) that promotes adaptive translational responses to stress, and oxidative phosphorylation (OXPHOS), the electron transport metabolic pathway that produces ATP. 4) Profiling of mRNAs bound to polysomes in RpL17-EC revealed increased translational efficiency (TE) of selective redox regulators, pro-inflammatory mediators, matrix metalloproteases, and endothelial to mesenchymal transition (endMT) factors; consistent with processes that promote IMT. 5) Increased levels of RNA binding proteins (RBPs) in EC-RpL17, such as QKI, HuR, and hn-RNP, correlated with enhanced stability of specific mRNAs and their TE. 6) Increased TE of glycolytic enzymes and the lactate transporter was consistent with increased EC glycolysis and OXPHOS dysfunction. 7) Increased glycolysis correlated with greater cell proliferation, suggesting RpL17 is a growth suppressor. Conclusion: These data define a new mechanism for control of EC function via selective changes in mRNA stability and ribosome profile and number that decreased OXPHOS and global protein translation, leading to EC-proliferation and endMT, which enhanced IMT in the PCL model of disturbed flow.

Abstract 321: Endothelial Cell Translational Control Is A Key Mediator Of Intima Growth In Response To Disturbed Flow

Background: Carotid intima-media thickening (IMT) is a good surrogate for atherosclerosis and CVD. Previously we used the IMT response of inbred mouse strains to disturbed flow (d-flow) in the partial carotid ligation (PCL) model to identify an intima modifier locus Im3. Microarray and QTL analysis revealed ribosomal protein large subunit (RpL17) as the primary candidate gene for IMT within Im3; further, low RpL17 levels correlate with enhanced intima formation. RpL17 is located in the nascent protein exit tunnel, and serves as a docking site for the NAC chaperone in protein folding. We generated endothelial cell (EC)-specific conditional heterozygous RpL17 mice (EC-RpL17+/-, termed EC-RpL17) to study the IMT response to d-flow using PCL. Results: EC-RpL17 mice had increased IMT relative to controls (RpL17+/+) after PCL. Data showed induction of oxidative stress and endoplasmic reticulum (ER) stress in EC-RpL17 with activation of the integrated stress response (ISR), a stress-support pathway promoting hyperproliferation in cancer, including: 1) increased levels of reactive oxygen species (ROS), BiP, Chop, and Atf4 expression, activation of PERK (p-PERK), and inactivation of eIF2 alpha (p-eIF2 alpha) in cultured RpL17 microvascular EC. The in vitro data correlated well with in vivo data from post-PCL carotid immunostaining, including: increased p-PERK, BiP, Chop, 4-hydroxynonenal and 8-hydroxyguanosine. EM analysis revealed dilation of the rough ER (indicating ER stress), and decreased cytosolic polyribosomes in EC-RpL17 EC. Conclusion: Decreased EC RpL17 increases d-flow mediated IMT, likely due to induction of growth-promoting ISR by ER and oxidative stress. These data define a novel pathophysiological role for RpL17.