A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

Selection and validation of reference genes for normalization of gene expression in Floccularia luteovirens.

Floccularia luteovirens is one of the rare edible fungi with high nutritional value found on the Qinghai-Tibet Plateau. However, research at the molecular level on this species is currently constrained due to the lack of reliable reference genes for this species. Thirteen potential reference genes (ACT, GAPDH, EF-Tu, SAMDC, UBI, CLN1, ß-TUB, γ-TUB, GTP, H3, UBC, UBC-E2, and GTPBP1) were chosen for the present study, and their expression under various abiotic conditions was investigated. Stability of gene expression was tested using GeNorm, NormFinder, BestKeeper, Delta-Ct, and RefFinder. The results showed that the most suitable reference genes for salt treatment were ACT and EF-Tu. Under drought stress, γ-TUB and UBC-E2 would be suitable for normalization. Under oxidative stress, the reference genes H3 and GAPDH worked well. Under heat stress, the reference genes EF-Tu and γ-TUB were suggested. Under extreme pH stress, UBC-E2 and H3 were appropriate reference genes. Under cadmium stress, the reference genes ACT and UBC-E2 functioned well. In different tissues, H3 and GTPBP1 were appropriate reference genes. The optimal internal reference genes when analyzing all samples were H3 and SAMDC. The expression level of HSP90 was studied to further validate the applicability of the genes identified in this study.

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background.

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.IMPORTANCEThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Unlocking translational machinery for antitubercular drug development.

Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.

Elongation factor MdEF-Tu coordinates with heat shock protein MdHsp70 to enhance apple thermotolerance.

High-temperature stress results in protein misfolding/unfolding and subsequently promotes the accumulation of cytotoxic protein aggregates that can compromise cell survival. Heat shock proteins (HSPs) function as molecular chaperones that coordinate the refolding and degradation of aggregated proteins to mitigate the detrimental effects of high temperatures. However, the relationship between HSPs and protein aggregates in apples under high temperatures remains unclear. Here, we show that an apple (Malus domestica) chloroplast-localized, heat-sensitive elongation factor Tu (MdEF-Tu), positively regulates apple thermotolerance when it is overexpressed. Transgenic apple plants exhibited higher photosynthetic capacity and better integrity of chloroplasts during heat stress. Under high temperatures, MdEF-Tu formed insoluble aggregates accompanied by ubiquitination modifications. Furthermore, we identified a chaperone heat shock protein (MdHsp70), as an interacting protein of MdEF-Tu. Moreover, we observed obviously elevated MdHsp70 levels in 35S: MdEF-Tu apple plants that prevented the accumulation of ubiquitinated MdEF-Tu aggregates, which positively contributes to the thermotolerance of the transgenic plants. Overall, our results provide new insights into the molecular chaperone function of MdHsp70, which mediates the homeostasis of thermosensitive proteins under high temperatures.

Structural basis for the toxicity of Legionella pneumophila effector SidH.

Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA.

IMPORTANCE: Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Testosterone and estradiol modify the expression of adhesins and biofilm formation in Actinobacillus seminis.

Actinobacillus seminis is the causal agent of epididymitis and has other effects on the reproductive tracts of small ruminants and bovines. This bacterium causes infection when luteinizing (LH) or follicle-stimulating hormones increase, and hosts reach sexual maturity. LH induces female ovulation and male testosterone production, suggesting that these hormones affect A. seminis pathogenicity. In the present study, we evaluated the effect of testosterone (1-5 ng/ml) or estradiol (5-25 pg/ml) added to culture medium on the in vitro growth, biofilm production, and adhesin expression of A. seminis. Estradiol does not promote the growth of this bacterium, whereas testosterone increased A. seminis planktonic growth 2-fold. Both hormones induced the expression of the elongation factor thermo unstable (EF-Tu) and phosphoglycerate mutase (PGM), proteins that A. seminis uses as adhesins. Estradiol (5 or 10 pg/ml) decreased biofilm formation by 32%, whereas testosterone, even at 5 ng/ml, showed no effect. Both hormones modified the concentrations of carbohydrates and eDNA in biofilms by 50%. Amyloid proteins are characterized by their capacity to bind Congo red (CR) dye. Actinobacillus seminis binds CR dye, and this binding increases in the presence of 5-20 pg/ml estradiol or 4 ng/ml testosterone. The A. seminis EF-Tu protein was identified as amyloid-like protein (ALP). The effect of sexual hormones on the growth and expression of virulence factors of A. seminis seems to be relevant for its colonization and permanence in the host.

Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli.

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10 fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

Sharing of Moonlighting Proteins Mediates the Symbiotic Relationship among Intestinal Commensals.

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called "moonlighting proteins," between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)-two well-known mucin-adhesive moonlighting proteins of B. longum-led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them.

Chloroplast translation factor EF-Tu of Arabidopsis thaliana can be inactivated via oxidation of a specific cysteine residue.

Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.

Detection of immunoreactive proteins of Escherichia coli, Streptococcus uberis, and Streptococcus agalactiae isolated from cows with diagnosed mastitis.

Introduction: Mastitis is a widespread mammary gland disease of dairy cows that causes severe economic losses to dairy farms. Mastitis can be caused by bacteria, fungi, and algae. The most common species isolated from infected milk are, among others, Streptococcus spp., and Escherichia coli. The aim of our study was protein detection based on both in silico and in vitro methods, which allowed the identification of immunoreactive proteins representative of the following species: Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Methods: The study group included 22 milk samples and 13 serum samples obtained from cows with diagnosed mastitis, whereas the control group constituted 12 milk samples and 12 serum samples isolated from healthy animals. Detection of immunoreactive proteins was done by immunoblotting, while amino acid sequences from investigated proteins were determined by MALDI-TOF. Then, bioinformatic analyses were performed on detected species specific proteins in order to investigate their immunoreactivity. Results: As a result, we identified 13 proteins: 3 (molybdenum cofactor biosynthesis protein B, aldehyde reductase YahK, outer membrane protein A) for E. coli, 4 (elongation factor Tu, tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG, GTPase Obg, glyceraldehyde-3-phosphate dehydrogenase) for S. uberis, and 6 (aspartate carbamoyltransferase, elongation factor Tu, 60 kDa chaperonin, elongation factor G, galactose-6-phosphate isomerase subunit LacA, adenosine deaminase) for S. agalactiae, which demonstrated immunoreactivity to antibodies present in serum from cows with diagnosed mastitis. Discussion: Due to the confirmed immunoreactivity, specificity and localization in the bacterial cell, these proteins can be considered considered potential targets in innovative rapid immunodiagnostic assays for bovine mastitis, however due to the limited number of examined samples, further examination is needed.

Ribosomal incorporation of negatively charged d-α- and N-methyl-l-α-amino acids enhanced by EF-Sep.

Ribosomal incorporation of d-α-amino acids (dAA) and N-methyl-l-α-amino acids (MeAA) with negatively charged sidechains, such as d-Asp, d-Glu, MeAsp and MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNAPro1E2, whose T-stem is derived from Escherichia coli tRNAGlu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, MeAsp and MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or MeAsp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Cell surface-associated protein elongation factor Tu interacts with fibronectin mediating the adhesion of Lactobacillus plantarum HC-2 to Penaeus vannamei intestinal epithelium and inhibiting the apoptosis induced by LPS and pathogen in Caco-2 cells.

The adherent colonization of lactic acid bacteria in the animal intestine is the basis for their probiotic effect, and their bacteria surface proteins play an important role in this process. Previous work has demonstrated that Lactobacillus plantarum HC-2 can adhere and colonize the intestine of Penaeus vannamei, modulate the intestinal immune response and microbial diversity, protect the intestinal tissues from pathogenic damage, and improve the protection rate of shrimp. The aim of this work was to identify adhesion molecules on the surface of HC-2 and its adhesion receptors in the intestinal epithelium of shrimp. The elongation factor Tu (EF-Tu) on the surface of HC-2 was found to interact with Fibronectin (Fib) in the shrimp intestine by immunoblotting and yeast two-hybrid assays, and this interaction relationship was verified by immunoprecipitation (Co-IP). The adhesion of HC-2 to Caco-2 cells could be blocked via EF-Tu antibody confinement, and the adhesion of Fib to HC-2 could be blocked by Fib antibody confinement. Expression of Fib on the surface of HEK293T cells revealed a significant increase in the adhesion rate of HC-2 to HEK293T cells. Using immunofluorescence, a significant reduction in HC-2 adhesion to the intestine of shrimp was observed after blocking the Fib site in the shrimp intestine, particularly in Vibrio parahaemolyticus E1-infected intestines. In addition, the recombinant protein rEF-Tu was found to promote the growth of Caco-2 cells in a certain concentration range and significantly inhibit the apoptosis induced by LPS, Staphylococcus aureus and V. parahaemolyticus E1. Our results indicate that EF-Tu might participate in gut immunity and homeostasis, through its binding to the shrimp intestinal cells and inhibiting apoptosis.

Engineered Agrobacterium improves transformation by mitigating plant immunity detection.

Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

The Valsa Mali effector Vm1G-1794 protects the aggregated MdEF-Tu from autophagic degradation to promote infection in apple.

Macroautophagy/autophagy is a conserved degradation pathway in eukaryotes that is required for recycling unwanted intracellular components, maintaining homeostasis, and coping with biotic and abiotic stresses. Pathogens have evolved to subvert autophagic machinery by secreting host cell-entering effector proteins. Here, we provided evidence that an apple autophagy-related gene MdATG8i, activated autophagy and contributed to resistance against Valsa canker caused by Valsa Mali (Vm) when being overexpressed in apple. MdATG8i interacted with a plastid elongation factor Tu (MdEF-Tu) which became insoluble and aggregated during Vm infection and was degraded through the autophagy pathway. Intriguingly, we identified a highly-induced effector secreted from Vm, Vm1G-1794, which competitively interacted with MdATG8i, suppressed autophagy, and depleted MdEF-Tu out of MdATG8i complexes. The formation of stable MdEF-Tu aggregates caused by Vm1G-1794 promoted the susceptibility of apple to Vm. Overall, our study demonstrated that MdATG8i contributed to Vm resistance by targeting and degrading MdEF-Tu, and Vm1G-1794 competed with MdEF-Tu to target MdATG8i and prevent MdEF-Tu degradation, thus favoring infection.Abbreviations: 35S: cauliflower mosaic virus 35S promoter; AIM: ATG8-interacting motif; ATG8-PE: ATG8 conjugated with phosphatidylethanolamine; BiFC: biomolecular fluorescence complementation; Con A: concanamycin A; Co-IP: co-immunoprecipitation; DEPs: differentially expressed proteins; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; hpt: hours post-treatment; LCI: luciferase complementation imaging; MdATG8i: autophagy-related protein 8i in Malus domestica; MDC: monodansylcadaverine; MdEF-Tu: elongation factor Tu in Malus domestica; MdNBR1: neighbor of BRCA1 in Malus domestica; N. benthamiana: Nicotiana benthamiana; OE: overexpression; PAMP: pathogen-associated molecular pattern; PTI: pattern-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Ub: ubiquitin; V. Mali: Valsa Mali; WT: wild-type plant; YFP: yellow fluorescent protein.

Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases.

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop.

Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+.