Ribosomal Subunit Research Articles

Structural analysis of the ribosome assembly factor Nep1, an N1-specific pseudouridine methyltransferase, reveals mechanistic insights.

Nucleolar essential protein 1 (Nep1; also known as ribosomal RNA small subunit methyltransferase Nep1) is a crucial factor in forming small ribosomal subunits in eukaryotes and archaea. Nep1 possesses an S-adenosyl-L-methionine (SAM)-dependent SpoU-TrmD (SPOUT) ribosomal RNA (rRNA) methyltransferase (MTase) fold and catalyzes pseudouridine (Ψ) methylation at specific sites of the small subunit (SSU) rRNA. Mutations in Nep1 proteins result in a severe developmental disorder in humans and reduced growth in yeast, suggesting its role in ribosome biogenesis. In this study, the crystal structures of Nep1 from the archaebacterium Pyrococcus horikoshii (PhNep1), both in its apo and holo (adenosine or 5-methylthioadenosine bound) forms have been reported. The structural analysis of PhNep1 revealed an α/β fold featuring a deep trefoil knot akin to the SPOUT domain, with two novel extensions-a globular loop and a β-α-β extension. Moreover, the cofactor-binding site of PhNep1 exhibits a preformed pocket, topologically similar to that of other SPOUT-class MTases. Further, structural analysis of PhNep1 revealed that it forms a homodimer coordinated by inter-subunit hydrogen bonds and hydrophobic interactions. Moreover, the results of this study indicate that PhNep1 can specifically methylate consensus RNAs, having a pseudouridine (ψ) located at position 926 of helix 35 (h35) of 16S rRNA in P. horikoshii. The stability of the Nep1-RNA complex seems to be primarily assisted by the conserved arginine residues located at the dimeric interface.

The assembly factor Reh1 is released from the ribosome during its initial round of translation

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel of the pre-60S subunit. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation. Using cryo-electron microscopy to image Reh1-bound 80S ribosomes, we found they contain A site peptidyl tRNA, P site tRNA and eIF5A, indicating that Reh1 does not dissociate from 60S until translation elongation. We propose that Reh1 is displaced by the elongating peptide chain, making it the last assembly factor released from the nascent 60S subunit during its initial round of translation.

A RE-EVALUATION OF ZYGOCOTYLE (DIGENEA, PARAMPHISTOMOIDEA) BASED ON NEW GENETIC DATA SUPPORTS ITS SYNONYMIZATION WITH WARDIUS.

The Zygocotylidae Ward, 1917 is a compact family of amphistome digeneans that until now comprised 2 genera, each represented by a single species: Zygocotyle lunata (Diesing, 1836) Stunkard, 1916 and Wardius zibethicus Barker and East, 1915 in Barker 1915. Despite highly similar morphology, these genera are separated based on the presence (Zygocotyle) or absence (Wardius) of posterolateral projections (=lappets) on the ventral sucker and esophageal bulb. In the present study, we generated partial large ribosomal subunit (28S), internal transcribed spacer 2 (ITS2) rDNA, and cytochrome c oxidase subunit 1 (COI) sequences of Z. lunata from several hosts (12 avian and 1 rodent species) throughout North and South America as well as 28S and COI sequences of W. zibethicus from muskrat in North America. The newly generated 28S sequences were used for sequence comparison and phylogenetic analysis. The COI sequences were used for species-level comparisons. Our analysis revealed a close relationship and high sequence similarity between Z. lunata and W. zibethicus. Considering the low morphological and genetic differences, we synonymize ZygocotyleStunkard, 1916 with Wardius Barker and East, 1915 in Barker 1915 and transfer Z. lunata to Wardius as Wardius lunatus (Diesing, 1836) n. comb.

Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit.

The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G and EF-Tu and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We find that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this activity is blocked in the absence of the SRL.

Phytochemical, antioxidant, and antimicrobial properties of Bellardia trixago methanol and ethanol extracts: insights from ADMET and molecular docking approaches

Abstract This study investigates the antioxidant, antimicrobial, and phytochemical properties of ethanol and methanol extracts derived from Bellardia trixago flowers alongside molecular docking and pharmacokinetic assessments of stigmasterol, a key bioactive compound. The antioxidant activities of the ethanol and methanol extracts were determined, with the methanol extract demonstrating higher activity, 0.370 ± 0.002 mg/mL, compared to the ethanol extract, 0.95 ± 0.09 mg/mL. The total phenolic content of the ethanol extract was 79.14 ± 2.30 mg GAE/g extract DW, and its flavonoid content was 244.31 ± 12.51 mg QE/g extract DW. The methanol extract contained a lower phenolic content of 51.50 ± 1.43 mg GAE/g extract DW but a higher flavonoid content of 251.67 ± 6.68 mg QE/g extract DW. The ethanol extract exhibited a superior iron chelation capacity of 8.21 ± 0.09 mg/mL relative to the methanol extract of 6.68 ± 0.28 mg/mL. Antimicrobial assays demonstrated that both extracts exhibited strong bactericidal and bacteriostatic effects, with MIC values ranging from < 0.39 mg/mL to > 125 mg/mL. The highest antimicrobial activity was observed against Bacillus cereus NRRL B-3711. Phytochemical analysis identified 25 compounds in the methanol extract and 20 in the ethanol extract. Stigmasterol was the major constituent in both extracts, accounting for 26.51% in ethanol and 12.65% in methanol. Molecular docking studies of stigmasterol revealed strong binding affinities with several protein targets, including Candida albicans Complex III2 (-8.7 kcal/mol), Escherichia coli HipBST (-7.2 kcal/mol), and the ribosomal subunit of Staphylococcus aureus (-7.0 kcal/mol). These interactions highlight stigmasterol’s potential role in modulating bacterial and fungal protein functions, emphasising the potential therapeutic applications of B. trixago, particularly in antimicrobial and antioxidant contexts.

The 40S ribosomal subunit recycling complex modulates mitochondrial dynamics and endoplasmic reticulum - mitochondria tethering at mitochondrial fission/fusion hotspots

The 40S ribosomal subunit recycling pathway is an integral link in the cellular quality control network, occurring after translational errors have been corrected by the ribosome-associated quality control (RQC) machinery. Despite our understanding of its role, the impact of translation quality control on cellular metabolism remains poorly understood. Here, we reveal a conserved role of the 40S ribosomal subunit recycling (USP10-G3BP1) complex in regulating mitochondrial dynamics and function. The complex binds to fission-fusion proteins located at mitochondrial hotspots, regulating the functional assembly of endoplasmic reticulum-mitochondria contact sites (ERMCSs). Furthermore, it alters the activity of mTORC1/2 pathways, suggesting a link between quality control and energy fluctuations. Effective communication is essential for resolving proteostasis-related stresses. Our study illustrates that the USP10-G3BP1 complex acts as a hub that interacts with various pathways to adapt to environmental stimuli promptly. It advances our molecular understanding of RQC regulation and helps explain the pathogenesis of human proteostasis and mitochondrial dysfunction diseases.

New Henneguya Species Cause Gill Disease of Commercial Amazonian Fish.

Freshwater fish are affected with much parasitic diseases, among the most common are Henneguyosis caused by myxozoans of the genus Henneguya, which exhibit great diversity in fish from South America, particularly in the Brazilian Amazon. In this present study, we describe the morphological and phylogenetic aspects of the small ribosomal subunit (SSU rDNA) of two new species of Henneguya infecting the gills from Hypophthalmus marginatus, a freshwater catfish from the Amazon. In 148 specimens, has been observed cyst formation in different regions of the gills, intrafilamentary and intralamellar. These samples were collected for PCR amplification and phylogenetic analysis. The myxospores from each cyst have an elliptical spore body, consisting of two elliptical polar capsules, but differed in taxonomic morphometric measurements, such as total size, caudal length, spore body length and width, and polar capsule length and width. Phylogenetically, the species formed a clade with Henneguya spp. described in Siluriform fish in Brazil. These findings support the description of two species, Henneguya matosi n. sp. and H. marginatus n. sp., which infect different regions of the gills of Hypophthalmus marginatus, a commercially important catfish.

Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection.

In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3'-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine-Dalgarno (SD) sequence in the mRNA and the anti-Shine-Dalgarno (ASD) sequence in 16S rRNA, to create "orthogonal" ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling the mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here, we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have an improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of binding of bS1 to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes.

Yeast Eukaryotic Initiation Factor 4B Remodels the MRNA Entry Site on the Small Ribosomal Subunit.

Eukaryotic Initiation Factor 4 (eIF4) is a group of factors that activates mRNA for translation and recruit 43S preinitiation complex (PIC) to the mRNA 5' end, forming the 48S PIC. The eIF4 factors include mRNA 5' cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffold protein eIF4G, which anchors eIF4A and eIF4E. Another eIF4 factor, eIF4B, stimulates the RNA helicase activity of eIF4A and facilitates mRNA recruitment. However, the mechanisms by which eIF4B binds the 40S ribosomal subunit and promotes mRNA recruitment remain poorly understood. Using cryo-Eletron Microscopy (cryo-EM), we obtained a map of the yeast 40S ribosomal subunit in a complex with eIF4B (40S-eIF4B complex). An extra density, tentatively assigned to yeast eIF4B, was observed near the mRNA entry channel of the 40S, contacting ribosomal proteins uS10, uS3, and eS10 as well as rRNA helix h16. Predictive modeling of the 40S-eIF4B complex suggests that the N-terminal domain of eIF4B binds near the mRNA entry channel, overlapping with the extra density observed in the 40S-eIF4B map. The partially open conformation of 40S in the 40S-eIF4B map is incompatible with eIF3j binding observed in the 48S PIC. Additionally, the extra density at the mRNA entry channel poses steric hindrance for eIF3g binding in the 48S PIC. Thus, structural insights suggest that eIF4B facilitates the release of eIF3j and the relocation of the eIF3b-g-i module during mRNA recruitment, thereby advancing our understanding of eIF4B's role in translation initiation.

RNA Sequencing Dataset of Drosophila Nociceptor Translatomic Response to Injury

To prepare to address the mechanisms of injury-induced nociceptor sensitization, we sequenced the translatome of the nociceptors of injured Drososophila larvae and those of uninjured larvae. Third-instar larvae expressing a green fluorescent protein (GFP)-tagged ribosomal subunit specifically in Class 4 dendritic arborization neurons, recognized as pickpocket-expressing primary nociceptors, via the GAL4/UAS method, were injured by ultraviolet light or sham-injured. Larvae were subjected to translating ribosome affinity purification for the GFP tag and nociceptor-specific ribosome-bound RNA was sequenced.

A new statistical metric for robust target detection in cryo-EM using 2D template matching.

2D template matching (2DTM) can be used to detect molecules and their assemblies in cellular cryo-EM images with high positional and orientational accuracy. While 2DTM successfully detects spherical targets such as large ribosomal subunits, challenges remain in detecting smaller and more aspherical targets in various environments. In this work, a novel 2DTM metric, referred to as the 2DTM p-value, is developed to extend the 2DTM framework to more complex applications. The 2DTM p-value combines information from two previously used 2DTM metrics, namely the 2DTM signal-to-noise ratio (SNR) and z-score, which are derived from the cross-correlation coefficient between the target and the template. The 2DTM p-value demonstrates robust detection accuracies under various imaging and sample conditions and outperforms the 2DTM SNR and z-score alone. Specifically, the 2DTM p-value improves the detection of aspherical targets such as a modified artificial tubulin patch particle (500 kDa) and a much smaller clathrin monomer (193 kDa) in simulated data. It also accurately recovers mature 60S ribosomes in yeast lamellae samples, even under conditions of increased Gaussian noise. The new metric will enable the detection of a wider variety of targets in both purified and cellular samples through 2DTM.

Unveiling the role of microRNAs in nonhost resistance to Sclerotinia sclerotiorum: Rice-specific microRNAs attack the pathogen via cross-kingdom RNAi.

The development of rapeseed with high resistance against the pathogen Sclerotinia sclerotiorum is impeded by the lack of effective resistance resources within host species. Unraveling the molecular basis of nonhost resistance (NHR) holds substantial value for resistance improvement in crops. In the present study, small RNA sequencing and transcriptome sequencing were carried out between rice (a nonhost species of S. sclerotiorum) and rapeseed during infection, revealing the involvement of rice miRNAs on translation-related processes in both rice and the pathogen. Specifically, rice-specific miRNAs with potential capability for cross-kingdom RNAi against S. sclerotiorum were explored, of which Os-miR169y was selected as a representative case to elucidate its role in resistance to S. sclerotiorum. The silence of Os-miR169y decreased the resistance level of rice to S. sclerotiorum, and heterologous expression of Os-miR169y in Arabidopsis and rapeseed significantly enhanced the host resistance. The dual-luciferase reporter assay indicates that Os-miR169y targets S. sclerotiorum 60S ribosomal protein L19 (SsRPL19). Overexpressing Os-miR169y (OEss-miR169y) and RNAi of SsRPL19 (RNAiss-RPL19) in S. sclerotiorum significantly impaired the growth and pathogenicity of the pathogen, while overexpressing SsRPL19 exhibited a contrast effect. Yeast-two-hybridization revealed an interlinking role of SsRPL19 with multiple large and small ribosomal subunits, indicating its important role in translation. Proteome sequencing detected a decreased amount of proteins in transformants OEss-miR169y and RNAiss-RPL19 and significant suppression on key metabolic pathways such as carbon and nitrogen metabolisms. Collectively, this study suggests that rice can secrete specific miRNAs to suppress genes essential for S. sclerotiorum, such as Os-miR169y, which targets and suppresses SsRPL19 and thus impairs protein synthesis in the pathogen. This study sheds light on the intrinsic mechanisms of rice NHR against S. sclerotiorum, and further demonstrates the potential of using nonhost-specific "pathogen-attacking" miRNAs in improving resistance in host species.

Kidney Targeting Smart Antibiotic Discovery: Multimechanism Pleuromutilins for Pyelonephritis Therapy.

Multidrug-resistant (MDR) bacteria pose a global health threat, underscoring the need for new antibiotics. Lefamulin, the first novel-mechanism antibiotic approved by the FDA in decades, showcases pleuromutilins' promise due to low mutation frequency. However, their clinical use is limited by poor pharmacokinetics and organ toxicity. To overcome these limitations, we modified lefamulin's C14 side chain via quaternization and incorporated rigid molecular fragments to enhance pharmacological properties. Introducing a quaternary ammonium group improved liver and kidney targeting via organic cation transporters (OCTs). Candidate 8i, a quaternized imidazo[4,5-c]pyridine pleuromutilin, demonstrated broad-spectrum activity against MDR bacteria, Mycoplasma and Chlamydophila at low doses. 8i targeted transport to infected kidneys, disrupted biofilms, damaged membranes, and inhibited protein synthesis by targeting 50S ribosomal subunit. It cleared rapidly, reducing long-term toxicity. Daily injections were an effective short-course treatment for systemic infections and pyelonephritis. This research presents a novel OCT-mediated, organ-targeted antibiotic design strategy to manage antibiotic-resistant infections.

Circ_0004662 contributes to colorectal cancer progression by interacting with hnRNPM.

Circular (circ)RNAs participate in colorectal cancer (CRC) occurrence and progression. However, the role of hsa_circ_0004662 (circ_0004662) in CRC remains unknown. Reverse transcription‑quantitative PCR noted high expression of circ_0004662 in CRC compared with normal colorectal epithelial cells. circ_0004662 knockdown inhibited migration of CRC cells in vitro and in vivo; would healing and Transwell assays showed that circ_0004662 overexpression contributed to CRC migration. Nuclear cytoplasmic analysis and fluorescence in situ hybridization revealed localization of circ_0004662 in the nucleus and cytoplasm. CircRNADB databases predicted that circ_0004662 exhibited translational potential and liquid chromatography‑mass spectrometry (LC‑MS) of circ_0004662 pull‑down products suggested that circ_0004662 bound to multiple ribosomal subunits. However, peptide products of 149aa translated by circ_0004662, with a molecular weight of ~17 kDa were not detected. Nevertheless, LC‑MS analysis indicated that circ_0004662 bound multiple proteins. Immunoprecipitation of RNA‑binding proteins revealed that circ_0004662 bound to heterogeneous nuclear ribonucleoprotein M (hnRNPM) and that hnRNPM interference decreased circ_0004662 expression, thereby affecting CRC progression. In summary, circ_0004662 was significantly upregulated in CRC. As a non‑coding RNA, it may promote CRC progression by binding to hnRNPM, which may serve as a potential target for treating CRC.

TP53 mutations and MDM2 polymorphisms in breast and ovarian cancers: amelioration by drugs and natural compounds.

Globally, breast and ovarian cancers are major health concerns in women and account for significantly high cancer-related mortality rates. Dysregulations and mutations in genes like TP53, BRCA1/2, KRAS and PTEN increase susceptibility towards cancer. Here, we discuss the impact of mutations in the key regulatory gene, TP53 and polymorphisms in its negative regulator MDM2 which are reported to accelerate cancer progression. Missense mutations, null mutations, transversions, transitions, and point mutations occurring in the TP53 gene can cause an increase in metastatic activity. This review discusses mutations occurring in exon regions of TP53, polymorphisms in MDM2 and their interaction with large ribosomal subunit protein (RPL) leading to cancer development. We also highlight the potential of small molecules e.g. p53 activators like XI-011, Tenovin-1, and Nutlin-3a for the treatment of breast and ovarian cancers. The therapeutic efficacy of natural compounds in amelioration of these two types of cancers is also discussed.

Novel archaeal ribosome dimerization factor facilitating unique 30S-30S dimerization.

Protein synthesis (translation) consumes a substantial proportion of cellular resources, prompting specialized mechanisms to reduce translation under adverse conditions. Ribosome inactivation often involves ribosome-interacting proteins. In both bacteria and eukaryotes, various ribosome-interacting proteins facilitate ribosome dimerization or hibernation, and/or prevent ribosomal subunits from associating, enabling the organisms to adapt to stress. Despite extensive studies on bacteria and eukaryotes, understanding factor-mediated ribosome dimerization or anti-association in archaea remains elusive. Here, we present cryo-electron microscopy structures of an archaeal 30S dimer complexed with an archaeal ribosome dimerization factor (designated aRDF), from Pyrococcus furiosus, resolved at a resolution of 3.2 Å. The complex features two 30S subunits stabilized by aRDF homodimers in a unique head-to-body architecture, which differs from the disome architecture observed during hibernation in bacteria and eukaryotes. aRDF interacts directly with eS32 ribosomal protein, which is essential for subunit association. The binding mode of aRDF elucidates its anti-association properties, which prevent the assembly of archaeal 70S ribosomes.

The proline-rich antimicrobial peptide Api137 disrupts large ribosomal subunit assembly and induces misfolding

The proline-rich antimicrobial designer peptide Api137 inhibits protein expression in bacteria by binding simultaneously to the ribosomal polypeptide exit tunnel and the release factor (RF), depleting the cellular RF pool and leading to ribosomal arrest at stop codons. This study investigates the additional effect of Api137 on the assembly of ribosomes using an Escherichia coli reporter strain expressing one ribosomal protein per 30S and 50S subunit tagged with mCherry and EGFP, respectively. Separation of cellular extracts derived from cells exposed to Api137 in a sucrose gradient reveals elevated levels of partially assembled and not fully matured precursors of the 50S subunit (pre-50S). High-resolution structures obtained by cryogenic electron microscopy demonstrate that a large proportion of pre-50S states are missing up to five proteins (uL22, bL32, uL29, bL23, and uL16) and have misfolded helices in 23S rRNA domain IV. These data suggest a second mechanism for Api137, wherein it disrupts 50S subunit assembly by inducing the formation of misfolded precursor particles potentially incapable of evolving into active ribosomes, suggesting a bactericidal mechanism.

Proteomic analysis reveals the difference between the spermatozoa of young and old Sus scrofa

The Wannan black pig is a superior local breed in Anhui province, renowned for its exceptional meat quality and remarkable adaptability to various environmental conditions. Semen, being a crucial indicator of male sexual maturity and fertility, significantly influences the performance of breeding boars. The molecular basis for comprehending the fecundity of boars in practical production lies in understanding the disparities in sperm proteins among boars of varying ages. In this investigation, spermatozoa from three one-year-old and three seven-year-old Wannan black pigs were individually chosen. Employing a tandem mass tag (TMT)-based quantitative proteomics approach, a total of 4050 proteins were identified, out of which 130 proteins exhibited significant differences between the two groups. GO enrichment analysis revealed that these proteins primarily participated in energy metabolism, spermatogenesis, fertilization, and reproduction. KEGG enrichment analysis demonstrated that the differential proteins predominantly resided within the ribosome pathway. A protein-protein interaction (PPI) network was constructed to identify core proteins such as Small ribosomal subunit protein uS7 (RPS5). Ultimately, parallel reaction monitoring (PRM) was conducted on the selected differential proteins to validate result accuracy. The findings of this study establish a foundation for elucidating the molecular mechanism underlying variations in spermatozoa protein levels among Wannan Black Pig with different age.

Autonomous ribosome biogenesis in vitro

Ribosome biogenesis is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs and 54 ribosomal proteins are synthesized and subjected to cooperative hierarchical assembly facilitated by numerous accessory factors. Realizing ribosome biogenesis in vitro is a critical milestone for understanding the self-replication of life and creating artificial cells. Despite its importance, this goal has not yet been achieved owing to its complexity. In this study, we report the successful realization of ribosome biogenesis in vitro. Specifically, we developed a highly specific and sensitive reporter assay for the detection of nascent ribosomes. The reporter assay allowed for combinatorial and iterative exploration of reaction conditions for ribosome biogenesis, leading to the simultaneous, autonomous synthesis of both small and large subunits of ribosomes in vitro through transcription, translation, processing, and assembly in a single reaction space. Our achievement represents a crucial advancement toward revealing the fundamental principles underlying the self-replication of life and creating artificial cells.

Harnessing marine bacteria for next-gen antibiotics: potent inhibition of S. aureus and Riemerella anatipestifer through in vitro, omics, and chemoinformatics approach with enhanced production of secondary metabolites through zinc sulfate

The rise of bacterial infections and increasing antibiotic resistance underscores an urgent need for new, effective antimicrobial agents with marine bacteria offering a unique and promising source for novel antibiotic compounds to combat persistent and emerging pathogens. In this research, five compounds were achieved from marine Streptomyces sp., C2-13, and their yield was enhanced with the addition of zinc sulfate at 0.5 mM. All compounds have been evaluated for their antibacterial activity against multiple pathogens, among which good activity was achieved against S. aureus, while potent activity was achieved against Riemerella anatipestifer with its IC50 value at 200 µm and bactericidal effect at 300 µm. Among all compounds, 4 was more active against both pathogens. A transcriptome analysis of active compound 4 showed its antibacterial effect on R. anatipestifer by inhibiting 30S and 50S ribosomal subunits, resistance mechanisms, and gliding motility proteins IX secretion system (T9SS) and interfering with protein translations process, secretion system, defense and resistance mechanisms, ultimately resulting in effective inhibition of normal bacterial growth and its motility. To investigate the anti-bacterial mechanism, all compounds were docked with two enzymes and TLR4 protein for predicting the vaccine construct, and the best docking score was achieved against RMFP with the highest score of -12.9 for compound 4. In silico cloning was carried out to ensure the expression of proteins generated and were cloned using E. coli as a host. The simulation studies have shown that both compound 4–RMFP and TLR4–RMFP complex are stable with the system. To the best of our knowledge, this is the first study investigating marine bacterial metabolites against R. anatipestifer with their anti-bacterial mechanism and enhancing their yield through the addition of zinc sulfate ions.