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Electrodeposited chromium plating continues to be widely used in a number of specialized areas, such as weapons, transport, aerospace, etc. However, the formation of texture, hydrogen content and residual stress can degrade the serviceability and lead to material failure. The effect of post heat treatment processes on the relationship of texture, hydrogen content, residual stress and corrosion resistance of hexavalent [Cr(VI)] chromium coatings deposited on Cr-Ni-Mo-V steel substrates was investigated. Macrotexture was measured by XRD. Microtexture, dislocation density and grain size were studied by EBSD. With the increase of the heat treatment temperature, it was found that the fiber texture strength of the (222) plane tended to increase and subsequently decrease. Below 600 °C, the increase in the (222) plane texture carried a decrease in the hydrogen content, residual stress, microhardness and an increase in the corrosion resistance. In addition, crack density and texture strength were less affected by the heat treatment time. Notably, relatively fewer crack densities of 219/cm2, a lower corrosion current density of 1.798 × 10-6 A/dm2 and a higher microhardness of 865 HV were found under the preferred heat treatment temperature and time of 380 °C and 4 h, respectively. The hydrogen content and residual stress were 7.63 ppm and 61 MPa, with 86% and 75% reduction rates compared to the as-plated state, respectively. In conclusion, in our future judgement of the influence of heat treatment on coating properties, we can screen or determine to a certain extent whether the heat treatment process is reasonable or not by measuring only the macrotexture.
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The COVID-19 pandemic caused by SARS-CoV-2 has had a persistent and significant impact on global public health for 4 years. Recently, there has been a resurgence of seasonal influenza transmission worldwide. The co-circulation of SARS-CoV-2 and seasonal influenza viruses results in a dual burden on communities. Additionally, the pandemic potential of zoonotic influenza viruses, such as avian Influenza A/H5N1 and A/H7N9, remains a concern. Therefore, a combined vaccine against all these respiratory diseases is in urgent need. mRNA vaccines, with their superior efficacy, speed in development, flexibility, and cost-effectiveness, offer a promising solution for such infectious diseases and potential future pandemics. In this study, we present FLUCOV-10, a novel 10-valent mRNA vaccine created from our proven platform. This vaccine encodes hemagglutinin (HA) proteins from four seasonal influenza viruses and two avian influenza viruses with pandemic potential, as well as spike proteins from four SARS-CoV-2 variants. A two-dose immunization with the FLUCOV-10 elicited robust immune responses in mice, producing IgG antibodies, neutralizing antibodies, and antigen-specific cellular immune responses against all the vaccine-matched viruses of influenza and SARS-CoV-2. Remarkably, the FLUCOV-10 immunization provided complete protection in mouse models against both homologous and heterologous strains of influenza and SARS-CoV-2. These results highlight the potential of FLUCOV-10 as an effective vaccine candidate for the prevention of influenza and COVID-19.IMPORTANCEAmidst the ongoing and emerging respiratory viral threats, particularly the concurrent and sequential spread of SARS-CoV-2 and influenza, our research introduces FLUCOV-10. This novel mRNA-based combination vaccine, designed to counteract both influenza and COVID-19, by incorporating genes for surface glycoproteins from various influenza viruses and SARS-CoV-2 variants. This combination vaccine was highly effective in preclinical trials, generating strong immune responses and ensuring protection against both matching and heterologous strains of influenza viruses and SARS-CoV-2. FLUCOV-10 represents a significant step forward in our ability to address respiratory viral threats, showcasing potential as a singular, adaptable vaccine solution for global health challenges.
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Vacunas contra la COVID-19 , COVID-19 , Vacunas contra la Influenza , SARS-CoV-2 , Vacunas de ARNm , Animales , COVID-19/prevención & control , COVID-19/inmunología , Ratones , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Humanos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/virología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/administración & dosificación , Ratones Endogámicos BALB C , Femenino , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Virus de la Influenza A/genéticaRESUMEN
Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.
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BACKGROUND: The behaviors and ontogeny of Aedes aegypti are closely related to the spread of diseases caused by dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV) viruses. During the life cycle, Ae. aegypti undergoes drastic morphological, metabolic, and functional changes triggered by gene regulation and other molecular mechanisms. Some essential regulatory factors that regulate insect ontogeny have been revealed in other species, but their roles are still poorly investigated in the mosquito. RESULTS: Our study identified 6 gene modules and their intramodular hub genes that were highly associated with the ontogeny of Ae. aegypti in the constructed network. Those modules were found to be enriched in functional roles related to cuticle development, ATP generation, digestion, immunity, pupation control, lectins, and spermatogenesis. Additionally, digestion-related pathways were activated in the larvae and adult females but suppressed in the pupae. The integrated proteinâprotein network also identified cilium-related genes. In addition, we verified that the 6 intramodular hub genes encoding proteins such as EcKinase regulating larval molt were only expressed in the larval stage. Quantitative RTâPCR of the intramodular hub genes gave similar results as the RNA-Seq expression profile, and most hub genes were ontogeny-specifically expressed. CONCLUSIONS: The constructed gene coexpression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. Ultimately, these findings will be key in identifying potential molecular targets for disease control.
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Aedes , Dengue , Fiebre Amarilla , Infección por el Virus Zika , Virus Zika , Masculino , Animales , Femenino , Fiebre Amarilla/genética , Virus Zika/genética , Redes Reguladoras de Genes , Mosquitos Vectores , Proteínas/genética , LarvaRESUMEN
Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has rapidly spread around the globe. With a substantial number of mutations in its Spike protein, the SARS-CoV-2 Omicron variant is prone to immune evasion and led to the reduced efficacy of approved vaccines. Thus, emerging variants have brought new challenges to the prevention of COVID-19 and updated vaccines are urgently needed to provide better protection against the Omicron variant or other highly mutated variants. Materials and methods: Here, we developed a novel bivalent mRNA vaccine, RBMRNA-405, comprising a 1:1 mix of mRNAs encoding both Delta-derived and Omicron-derived Spike proteins. We evaluated the immunogenicity of RBMRNA-405 in BALB/c mice and compared the antibody response and prophylactic efficacy induced by monovalent Delta or Omicron-specific vaccine with the bivalent RBMRNA-405 vaccine in the SARSCoV-2 variant challenge. Results: Results showed that the RBMRNA-405 vaccine could generate broader neutralizing antibody responses against both Wuhan-Hu-1 and other SARS-CoV-2 variants, including Delta, Omicron, Alpha, Beta, and Gamma. RBMRNA-405 efficiently blocked infectious viral replication and lung injury in both Omicron- and Delta-challenged K18-ACE2 mice. Conclusion: Our data suggest that RBMRNA-405 is a promising bivalent SARS-CoV-2 vaccine with broad-spectrum efficacy for further clinical development.
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Vacunas contra la COVID-19 , COVID-19 , Animales , Humanos , Ratones , SARS-CoV-2 , COVID-19/prevención & control , Ratones Endogámicos BALB C , ARN Mensajero , Vacunas Combinadas , Vacunas de ARNmRESUMEN
Migration from rhizosphere to rhizoplane is a key selecting process in root microbiome assembly, but not fully understood. Rhizobiales members are overrepresented in the core root microbiome of terrestrial plants, and here we report a genome-wide transposon-sequencing of rhizoplane fitness genes of beneficial Sinorhizobium fredii on wild soybean, cultivated soybean, rice, and maize. There were few genes involved in broad-host-range rhizoplane colonization. The fadL mutant lacking a fatty acid transporter exhibited high colonization rates, while mutations in exoFQP (encoding membrane proteins directing exopolysaccharide polymerization and secretion), but not those in exo genes essential for exopolysaccharide biosynthesis, led to severely impaired colonization rates. This variation was not explainable by their rhizosphere and rhizoplane survivability, and associated biofilm and exopolysaccharide production, but consistent with their migration ability toward rhizoplane, and associated surface motility and the mixture of quorum-sensing AHLs (N-acylated-L-homoserine lactones). Genetics and physiology evidences suggested that FadL mediated long-chain AHL uptake while ExoF mediated the secretion of short-chain AHLs which negatively affected long-chain AHL biosynthesis. The fadL and exoF mutants had elevated and depleted extracellular long-chain AHLs, respectively. A synthetic mixture of long-chain AHLs mimicking that of the fadL mutant can improve rhizobial surface motility. When this AHL mixture was spotted into rhizosphere, the migration toward roots and rhizoplane colonization of S. fredii were enhanced in a diffusible way. This work adds novel parts managing extracellular AHLs, which modulate bacterial migration toward rhizoplane. The FadL-ExoFQP system is conserved in Alphaproteobacteria and may shape the "home life" of diverse keystone rhizobacteria.
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Rhizobium , Bacterias/genética , Percepción de Quorum , Biopelículas , Ácidos Grasos , Acil-Butirolactonas/metabolismoRESUMEN
Recent advancements in the production, modification, and cellular delivery of RNA molecules facilitated the expansion of RNA-based therapeutics. The increasing understanding of RNA biology initiated a corresponding growth in RNA therapeutics. In this review, the general concepts of five classes of RNA-based therapeutics, including RNA interference-based therapies, antisense oligonucleotides, small activating RNA therapies, circular RNA therapies, and messenger RNA-based therapeutics, will be discussed. Moreover, we also provide an overview of RNA-based therapeutics that have already received regulatory approval or are currently being evaluated in clinical trials, along with challenges faced by these technologies. RNA-based drugs demonstrated positive clinical trial results and have the ability to address previously "undruggable" targets, which delivers great promise as a disruptive therapeutic technology to fulfill its full clinical potentiality.
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Oligonucleótidos Antisentido , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Interferencia de ARN , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , ARN Mensajero/genéticaRESUMEN
The distribution and abundance of transposable elements across the tree of life have significantly shaped the evolution of cellular organisms, but the underlying mechanisms shaping these ecological patterns remain elusive. Here we establish a "common garden" approach to study causal ecological interactions between a xenogeneic conditional lethal sacB gene and the community of transposable insertion sequences (ISs) in a multipartite prokaryote genome. Xenogeneic sacB of low, medium, or high GC content was individually inserted into three replicons of a model bacterium Sinorhizobium fredii, and exhibited replicon- and GC-dependent variation in genetic stability. This variation was largely attributable to multidimensional niche differentiation for IS community members. The transposition efficiency of major active ISs depended on the nucleoid-associated xenogeneic silencer MucR. Experimentally eliminating insertion activity of specific ISs by deleting MucR strongly demonstrated a dominant role of niche differentiation among ISs. This intracellular common garden approach in the experimental evolution context allows not only for evaluating genetic stability of natural and synthetic xenogeneic genes of different sequence signatures in host cells but also for tracking and testing causal relationships in unifying ecological principles in genome ecology.
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Elementos Transponibles de ADN , Genoma Bacteriano , Bacterias/genética , Células Procariotas , ReplicónRESUMEN
There is an urgent need for a broad-spectrum and protective vaccine due to the emergence and rapid spreading of more contagious SARS-CoV-2 strains. We report the development of RBMRNA-176, a pseudouridine (Ψ) nucleoside-modified mRNA-LNP vaccine encoding pre-fusion stabilized trimeric SARS-CoV-2 spike protein ectodomain, and evaluate its immunogenicity and protection against virus challenge in mice and nonhuman primates. A prime-boost immunization with RBMRNA-176 at intervals of 21 days resulted in high IgG titers (over 1:819,000 endpoint dilution) and a CD4+ Th1-biased immune response in mice. RBMRNA-176 vaccination induced pseudovirus-neutralizing antibodies with IC50 ranging from 1:1020 to 1:2894 against SARS-CoV-2 spike pseudotyped wild-type and variant viruses, including Alpha, Beta, Gamma, and Kappa. Moreover, significant control of viral replication and histopathology in lungs was observed in vaccinated mice. In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the prime induced a persistent and sustained IgG response. RBMRNA-176 vaccination also protected macaques against upper and lower respiratory tract infection, as well as lung injury. Altogether, these findings support RBMRNA-176 as a vaccine candidate for prevention of COVID-19.
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Bacterial adaptation is largely shaped by horizontal gene transfer, xenogeneic silencing mediated by lineage-specific DNA bridgers (H-NS, Lsr2, MvaT and Rok), and various anti-silencing mechanisms. No xenogeneic silencing DNA bridger is known for α-proteobacteria, from which mitochondria evolved. By investigating α-proteobacterium Sinorhizobium fredii, a facultative legume microsymbiont, here we report the conserved zinc-finger bearing MucR as a novel xenogeneic silencing DNA bridger. Self-association mediated by its N-terminal domain (NTD) is required for DNA-MucR-DNA bridging complex formation, maximizing MucR stability, transcriptional silencing, and efficient symbiosis in legume nodules. Essential roles of NTD, CTD (C-terminal DNA-binding domain), or full-length MucR in symbiosis can be replaced by non-homologous NTD, CTD, or full-length protein of H-NS from γ-proteobacterium Escherichia coli, while NTD rather than CTD of Lsr2 from Gram-positive Mycobacterium tuberculosis can replace the corresponding domain of MucR in symbiosis. Chromatin immunoprecipitation sequencing reveals similar recruitment profiles of H-NS, MucR and various functional chimeric xenogeneic silencers across the multipartite genome of S. fredii, i.e. preferring AT-rich genomic islands and symbiosis plasmid with key symbiosis genes as shared targets. Collectively, the convergently evolved DNA bridger MucR predisposed α-proteobacteria to integrate AT-rich foreign DNA including symbiosis genes, horizontal transfer of which is strongly selected in nature.
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Alphaproteobacteria , Regulación Bacteriana de la Expresión Génica , Alphaproteobacteria/genética , Proteínas Bacterianas/metabolismo , ADN , Escherichia coli/genética , Escherichia coli/metabolismo , SimbiosisRESUMEN
Foreign AT-rich genes drive bacterial adaptation to new niches while challenging the existing regulation network. Here we report that MucR, a conserved regulator in α-proteobacteria, balances adaptation and regulatory integrity in Sinorhizobium fredii, a facultative microsymbiont of legumes. Chromatin immunoprecipitation sequencing coupled with transcriptomic data reveal that average transcription levels of both target and non-target genes, under free-living and symbiotic conditions, increase with their conservation levels. Targets involved in environmental adaptation and symbiosis belong to genus or species core and can be repressed or activated by MucR in a condition-dependent manner, implying regulatory integrations. However, most targets are enriched in strain-specific genes of lower expression levels and higher AT%. Within each conservation levels, targets have higher AT% and average transcription levels than non-target genes and can be further up-regulated in the mucR mutant. This is consistent with higher AT% of spacers between -35 and -10 elements of promoters for target genes, which enhances transcription. The MucR recruitment level linearly increases with AT% and the number of a flexible pattern (with periodic repeats of Ts) of target sequences. Collectively, MucR directly represses AT-rich foreign genes with predisposed high transcription potential while progressive erosions of its target sites facilitate regulatory integrations of foreign genes.
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Alphaproteobacteria , Regulación Bacteriana de la Expresión Génica , Alphaproteobacteria/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simbiosis/genética , Zinc/metabolismoRESUMEN
The functional inactivation of tumor suppressor microRNA (miRNA) is closely related to the tumorigenesis of cancer. There are instances where the miRNA and the corresponding target both exist in a cell, but the target gene silencing do not occur as expected. Herein, we found that both miR-506 and its target CDK6 are highly co-expressed in lung cancer cells. Sequence analyses suggested that a miR-506 binding site (1648-1654) and a cis-element (1785-1795) for binding by heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) are evolutionarily conserved and forms a stem structure in the 3' untranslated region (3'UTR) of CDK6. Furthermore, HNRNPA2B1 can bind to the stem structure to denature it and recruit the RNA helicase DExH-box helicase 9 (DHX9) to the 3'UTR, which ultimately facilitates miRNAs-mediated CDK6 silencing. These results indicate that the cis-element of the 3'UTR of CDK6, where HNRNPA2B1 binds, serves as an RNA switch to regulate miRNAs' function in cancer cells.
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Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.
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Química Clic , ARN , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteómica , Análisis de Secuencia de ARNRESUMEN
The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.
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Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glycine max/microbiología , Liasas de Fósforo-Oxígeno/genética , Sinorhizobium/genética , Simbiosis/genética , Transcripción Genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/clasificación , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Fijación del Nitrógeno/genética , Liasas de Fósforo-Oxígeno/biosíntesis , Liasas de Fósforo-Oxígeno/clasificación , Liasas de Fósforo-Oxígeno/metabolismo , Sinorhizobium/fisiologíaRESUMEN
Prokaryotes harbor a various proportion of accessory genes in their genomes. The integration of accessory functions with the core regulation network is critical for environmental adaptation, particularly considering a theoretically unlimited number of niches on the earth for microorganisms. Comparative genomics can reveal a co-occurrence pattern between a subset of accessory genes (or variations in core genes) and an adaptation trait, while comparative transcriptomics can further uncover whether a coordinated regulation of gene expression is involved. In this chapter, we introduce a protocol for weighted gene coexpression network construction by using well-developed open source tools, and a further application of such a network in comparative analysis of bacterial core and accessory genes.
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ADN Bacteriano/genética , Redes Reguladoras de Genes , Genoma Bacteriano , Genómica , Sinorhizobium fredii/genética , Bases de Datos Genéticas , Regulación Bacteriana de la Expresión Génica , Filogenia , Proyectos de Investigación , Flujo de TrabajoRESUMEN
The interkingdom coevolution innovated the rhizobium-legume symbiosis. The application of this nitrogen-fixing system in sustainable agriculture is usually impeded by incompatible interactions between partners. However, the progressive evolution of rhizobium-legume compatibility remains elusive. In this work, deletions of rhcV encoding a structural component of the type three secretion system allow related Sinorhizobium strains to nodulate a previously incompatible soybean cultivar (Glycine max). These rhcV mutants show low to medium to high symbiotic efficiency on the same cultivated soybean while being indistinguishable on wild soybean plants (Glycine soja). The dual pantranscriptomics reveals nodule-specific activation of core symbiosis genes of Sinorhizobium and Glycine genes associated with genome duplication events along the chronogram. Unexpectedly, symbiotic efficiency is in line with lineage-dependent transcriptional profiles of core pathways which predate the diversification of Fabaceae and Sinorhizobium. This is supported by further physiological and biochemical experiments. Particularly, low-efficiency nodules show disordered antioxidant activity and low-energy status, which restrict nitrogen fixation activity. Collectively, the ancient core pathways play a crucial role in optimizing the function of later-evolved mutualistic arsenals in the rhizobium-legume coevolution.IMPORTANCE Significant roles of complex extracellular microbiota in environmental adaptation of eukaryotes in ever-changing circumstances have been revealed. Given the intracellular infection ability, facultative endosymbionts can be considered pioneers within complex extracellular microbiota and are ideal organisms for understanding the early stage of interkingdom adaptation. This work reveals that the later innovation of key symbiotic arsenals and the lineage-specific network rewiring in ancient core pathways, predating the divergence of legumes and rhizobia, underline the progressive evolution of rhizobium-legume compatibility. This insight not only is significant for improving the application benefits of rhizobial inoculants in sustainable agriculture but also advances our general understanding of the interkingdom coevolution which is theoretically explored by all host-microbiota interactions.
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Iron homeostasis is strictly regulated in cellular organisms. The Rhizobiales order enriched with symbiotic and pathogenic bacteria has evolved a lineage-specific regulator, RirA, responding to iron fluctuations. However, the regulatory role of RirA in bacterium-host interactions remains largely unknown. Here, we report that RirA is essential for mutualistic interactions of Sinorhizobium fredii with its legume hosts by repressing a gene cluster directing biosynthesis and transport of petrobactin siderophore. Genes encoding an inner membrane ABC transporter (fat) and the biosynthetic machinery (asb) of petrobactin siderophore are sporadically distributed in Gram-positive and Gram-negative bacteria. An outer membrane siderophore receptor gene (fprA) was naturally assembled with asb and fat, forming a long polycistron in S. fredii. An indigenous regulation cascade harboring an inner membrane protease (RseP), a sigma factor (FecI), and its anti-sigma protein (FecR) were involved in direct activation of the fprA-asb-fat polycistron. Operons harboring fecI and fprA-asb-fat, and those encoding the indigenous TonB-ExbB-ExbD complex delivering energy to the outer membrane transport activity, were directly repressed by RirA under iron-replete conditions. The rirA deletion led to upregulation of these operons and iron overload in nodules, impaired intracellular persistence, and symbiotic nitrogen fixation of rhizobia. Mutualistic defects of the rirA mutant can be rescued by blocking activities of this naturally "synthetic" circuit for siderophore biosynthesis and transport. These findings not only are significant for understanding iron homeostasis of mutualistic interactions but also provide insights into assembly and integration of foreign machineries for biosynthesis and transport of siderophores, horizontal transfer of which is selected in microbiota. IMPORTANCE Iron is a public good explored by both eukaryotes and prokaryotes. The abundant ferric form is insoluble under neutral and basic pH conditions, and many bacteria secrete siderophores forming soluble ferric siderophore complexes, which can be then taken up by specific receptors and transporters. Siderophore biosynthesis and uptake machineries can be horizontally transferred among bacteria in nature. Despite increasing attention on the importance of siderophores in host-microbiota interactions, the regulatory integration process of transferred siderophore biosynthesis and transport genes is poorly understood in an evolutionary context. By focusing on the mutualistic rhizobium-legume symbiosis, here, we report how a naturally synthetic foreign siderophore gene cluster was integrated with the rhizobial indigenous regulation cascade, which is essential for maintaining mutualistic interactions.
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Fabaceae , Rhizobium , Sinorhizobium , Sideróforos/metabolismo , Fabaceae/microbiología , Sinorhizobium/metabolismo , Simbiosis/genética , Antibacterianos , Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Hierro/metabolismo , Bacterias/metabolismo , Proteínas de Transporte de Membrana , Verduras , Rhizobium/metabolismoRESUMEN
Pathogenic bacteria need high-affinity zinc uptake systems to counteract the nutritional immunity exerted by infected hosts. However, our understanding of zinc homeostasis in mutualistic systems such as the rhizobium-legume symbiosis is limited. Here, we show that the conserved high-affinity zinc transporter ZnuABC and accessory transporter proteins (Zip1, Zip2, and c06450) made cumulative contributions to nodulation of the broad-host-range strain Sinorhizobium fredii CCBAU45436. Zur acted as a zinc-dependent repressor for the znuC-znuB-zur operon, znuA, and c06450 by binding to the associated Zur box, but did not regulate zip1 and zip2 ZnuABC was the major zinc transporter. Combined mutants lacking znuA and one of the three accessory genes had more severe defects in nodulation and growth under zinc starvation conditions than the znuA mutant, though rhizoplane colonization by these mutants was not impaired. In contrast to the elite strain CCBAU45436, more drastic symbiotic defects were observed for the znuA mutants of other Sinorhizobium strains, which lack at least one of the three accessory genes in their genomes and are characterized by their limited host range and geographical distribution. The znu-derived mutants showed a higher expression level of nod genes involved in Nod factor biosynthesis and a reduced expression of genes encoding a type three secretion system and its effector NopP, which can interfere with the host immune system. Application of exogenous zinc restored the nodulation ability of these znu-derived mutants. Therefore, the conserved ZnuABC and accessory components in the zinc starvation machinery play an important role in modulating symbiotic compatibility.IMPORTANCE The rhizobium-legume symbiosis contributes around 65% of biological nitrogen fixation in agriculture systems and is critical for sustainable agriculture by reducing the amount of chemical nitrogen fertilizer being used. Rhizobial inocula have been commercialized for more than 100 years, but the efficiency of inoculation can vary among legume cultivars, field sites, and years. These long-lasting challenging problems impede the establishment of a sustainable agriculture, particularly in developing countries. Here, we report that rhizobial zinc starvation machinery containing a conserved high-affinity zinc transporter and accessory components makes cumulative contributions to modulating rhizobial symbiotic compatibility. This work highlights a critical role of largely unexplored nutritional immunity in the rhizobium-legume symbiosis, which makes zinc starvation machinery an attractive target for improving rhizobial symbiotic compatibility.
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Fabaceae/microbiología , Genes Bacterianos/genética , Rhizobium/genética , Rhizobium/metabolismo , Simbiosis/genética , Simbiosis/fisiología , Zinc/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Operón , Nodulación de la Raíz de la Planta , Análisis de Secuencia de Proteína , Sinorhizobium/genética , Sinorhizobium/metabolismo , Inanición , Sistemas de Secreción Tipo IIIRESUMEN
Colorectal cancer is a complex disease driven by well-established mutations such as APC and other yet to be identified pathways. The GTPase Rab11 regulates endosomal protein trafficking, and previously we showed that loss of Rab11 caused intestinal inflammation and hyperplasia in mice and flies. To test the idea that loss of Rab11 may promote cancer progression, we have analyzed archival human patient tissues and observed that 51 out of 70 colon cancer tissues had lower Rab11 protein staining. By using the Drosophila midgut model, we have found that loss of Rab11 can lead to three changes that may relate to cancer progression. First is the disruption of enterocyte polarity based on staining of the FERM domain protein Coracle. Second is an increased proliferation due to an increased expression of the JAK-STAT pathway ligand Upd3. Third is an increased expression of ImpL2, which is an IGFBP7 homolog and can suppress metabolism. Furthermore, loss of Rab11 can act synergistically with the oncoprotein RasV12 to regulate these cancer-related phenotypes.