Synthetic Microbial Community Promotes Bacterial Communities Leading to Soil Multifunctionality in Desertified Land.

Soil desertification is an important challenge in global soil management, and effectively and stably restoring soil function is an urgent problem. Using synthetic microbial communities (SynComs) is a burgeoning microbial strategy aimed at enhancing soil nutrients through functional synergies among diverse microorganisms; nevertheless, their effectiveness in restoring desertified soils remains unknown. In this study, we conducted a two-year field experiment using a SynCom constructed by in situ probiotic bacteria and set up control, chemical fertilizer, and combined SynCom-chemical fertilizer (combined fertilizer) treatments to investigate the linkage between microbial communities and soil multifunctionality in the soil surface layer (0-10 cm). Both the bacterial and fungal communities differed the most under the combined fertilizer treatment compared to the control. The bacterial communities differed more under treatments of the SynCom than the chemical fertilizer, while the fungal communities differed more under the chemical fertilizer treatment than the SynCom treatment. Regarding soil function, the SynCom strengthened the correlation between enzyme activities and both bacterial communities and functional properties. pH and available potassium were the main influencing factors under the chemical fertilizer and combined fertilizer treatments. The beta-diversity of the bacterial communities was significantly correlated with soil multifunctionality. Random forest analyses showed that the SynCom significantly enhanced the bacterial communities, driving soil multifunctionality, and that some potential microbial taxa drove multiple nutrient cycles simultaneously. In summary, the SynCom effectively increased the abundance of most carbon, nitrogen, and phosphorus functional genes as well as soil enzyme activities. The bacterial community composition contributed significantly to soil multifunctionality. Hence, the development of novel microbial agents holds significant potential for improving soil functionality and managing desertification.

Comprehensive proteomic profiling of lung adenocarcinoma: development and validation of an innovative prognostic model.

Background: Lung adenocarcinoma (LUAD), a global leading cause of cancer deaths, remains inadequately addressed by current protein biomarkers. Our study focuses on developing a protein-based risk signature for improved prognosis of LUAD. Methods: We employed the least absolute shrinkage and selection operator (LASSO)-COX algorithm on The Cancer Genome Atlas database to construct a prognostic model incorporating six proteins (CD49B, UQCRC2, SMAD1, FOXM1, CD38, and KAP1). The model's performance was assessed using principal component, Kaplan-Meier (KM), and receiver operating characteristic (ROC) analysis, indicating strong predictive capability. The model stratifies LUAD patients into distinct risk groups, with further analysis revealing its potential as an independent prognostic factor. Additionally, we developed a predictive nomogram integrating clinicopathologic factors, aimed at assisting clinicians in survival prediction. Gene set enrichment analysis (GSEA) and examination of the tumor immune microenvironment were conducted, highlighting metabolic pathways in high-risk genes and immune-related pathways in low-risk genes, indicating varied immunotherapy sensitivity. Validation through immunohistochemistry from the Human Protein Atlas (HPA) database and immunofluorescence staining of clinical samples was performed, particularly focusing on CD38 expression. Results: Our six-protein model (CD49B, UQCRC2, SMAD1, FOXM1, CD38, KAP1) effectively categorized LUAD patients into high and low-risk groups, confirmed by principal component, KM, and ROC analyses. The model showed high predictive accuracy, with distinct survival differences between risk groups. Notably, CD38, traditionally seen as protective, was paradoxically associated with poor prognosis in LUAD, a finding supported by immunohistochemistry and immunofluorescence data. GSEA revealed that high-risk genes are enriched in metabolic pathways, while low-risk genes align with immune-related pathways, suggesting better immunotherapy response in the latter group. Conclusions: This study presented a novel prognostic protein model for LUAD, highlighting the CD38 expression paradox and enhancing our understanding of protein roles in lung cancer progression. It offered new clinical tools for prognosis prediction and provided assistance for future lung cancer pathogenesis research.

A pyocin-like T6SS effector mediates bacterial competition in Yersinia pseudotuberculosis.

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis.IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.

Classical and novel properties of Holliday junction resolvase SynRuvC from Synechocystis sp. PCC6803.

Cyanobacteria, which have a photoautotrophic lifestyle, are threatened by ultraviolet solar rays and the reactive oxygen species generated during photosynthesis. They can adapt to environmental conditions primarily because of their DNA damage response and repair mechanisms, notably an efficient homologous recombination repair system. However, research on double-strand break (DSB) repair pathways, including the Holliday junction (HJ) resolution process, in Synechocystis sp. PCC6803 is limited. Here, we report that SynRuvC from cyanobacteria Synechocystis sp. PCC6803 has classical HJ resolution activity. We investigated the structural specificity, sequence preference, and biochemical properties of SynRuvC. SynRuvC strongly preferred Mn2+ as a cofactor, and its cleavage site predominantly resides within the 5'-TG↓(G/A)-3' sequence. Interestingly, novel flap endonuclease and replication fork intermediate cleavage activities of SynRuvC were also determined, which distinguish it from other reported RuvCs. To explore the effect of SynRuvC on cell viability, we constructed a knockdown mutant and an overexpression strain of Synechocystis sp. PCC6803 (synruvCKD and synruvCOE) and assessed their survival under a variety of conditions. Knockdown of synruvC increased the sensitivity of cells to MMS, HU, and H2O2. The findings suggest that a novel RuvC family HJ resolvase SynRuvC is important in a variety of DNA repair processes and stress resistance in Synechocystis sp. PCC6803.

Streptomyces pratensis S10 inhibits spread of Fusarium graminearum invasive hyphae and toxisome formation in wheat plants.

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum, leads to severe economic losses worldwide. Effective management measures for controlling FHB are not available, due to a lack of resistant cultivars. Currently, the utilization of biological control is a promising approach that can be used to help manage FHB. Previous studies have confirmed that Streptomyces pratensis S10 harbors excellent inhibitory effects on F. graminearum. However, there is no information regarding invasive hyphae of F. graminearum are inhibited by S10. Thus, we investigated the effects of S10 on F. graminearum strain PH-1 hyphae extension, toxisome formation, and TRI5 gene expression on wheat plants via microscopic observation. The results showed that S10 effectively inhibited spread of F. graminearum hyphae along the rachis, restricting the infection of neighboring florets via the phloem. In the presence of S10, the hyphal growth is impeded by formation of dense cell wall thickenings in the rachis internode surrounding the F. graminearum infection site, avoiding cell plasmolysis and collapse. We further demonstrated that S10 largely prevented cell-to-cell invasion of fungal hyphae inside wheat coleoptiles using a constitutively green fluorescence protein-expressing F. graminearum strain, PH-1. Importantly, S. pratensis S10 inhibited toxisome formation and TRI5 gene expression in wheat plants during infection. Collectively, these findings indicated that S. pratensis S10 prevents spread of F. graminearum invasive hyphae via the rachis.

Analysis of Methylesterase Gene Family in Fragaria vesca Unveils Novel Insights into the Role of FvMES2 in Methyl Salicylate-Mediated Resistance against Strawberry Gray Mold.

Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis.

Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE: Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur's regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.

Pseudomonas aeruginosa outer membrane vesicle-packed sRNAs can enter host cells and regulate innate immune responses.

Bacterial outer membrane vesicles (OMVs) can package and deliver virulence factors into host cells, which is an important mechanism mediating host-pathogen interactions. It has been reported that small RNAs (sRNAs) can be packed into OMVs with varying relative abundance, which might affect the function and/or stability of host mRNAs. In this study, we used OptiPrep density gradient ultra-high-speed centrifugation to purify OMVs from Pseudomonas aeruginosa. Next, the sequences and abundance of sRNAs were detected by using Small RNA-Seq. In particular, sRNA4518698, sRNA2316613 and sRNA809738 were the three most abundant sRNAs in OMVs, which are all fragments of P. aeruginosa non-coding RNAs. sRNAs were shielded within the interior of OMVs and remained resistant to external RNase cleavage. The miRanda and RNAhybrid analysis demonstrated that those sRNAs could target a large number of host mRNAs, which were enriched in host immune responses by the functions of GO and KEGG enrichment. Experimentally, we demonstrated that the transfection of synthetic sRNA4518698, sRNA2316613, or sRNA809738 could reduce the expression of innate immune response genes in RAW264.7 cells. Together, we demonstrated that P. aeruginosa OMVs sRNAs can regulate innate immune responses. This study uncovered a mechanism in which the OMVs regulate host responses by transferring bacterial sRNAs.

Trojan horselike T6SS effector TepC mediates both interference competition and exploitative competition.

The type VI secretion system (T6SS) is a bacterial weapon capable of delivering antibacterial effectors to kill competing cells for interference competition, as well as secreting metal ion scavenging effectors to acquire essential micronutrients for exploitation competition. However, no T6SS effectors that can mediate both interference competition and exploitation competition have been reported. In this study, we identified a unique T6SS-1 effector in Yersinia pseudotuberculosis named TepC, which plays versatile roles in microbial communities. First, secreted TepC acts as a proteinaceous siderophore that binds to iron and mediates exploitative competition. Additionally, we discovered that TepC has DNase activity, which gives it both contact-dependent and contact-independent interference competition abilities. In conditions where iron is limited, the iron-loaded TepC is taken up by target cells expressing the outer membrane receptor TdsR. For kin cells encoding the cognate immunity protein TipC, TepC facilitates iron acquisition, and its toxic effects are neutralized. On the other hand, nonkin cells lacking TipC are enticed to uptake TepC and are killed by its DNase activity. Therefore, we have uncovered a T6SS effector, TepC, that functions like a "Trojan horse" by binding to iron ions to provide a valuable resource to kin cells, whereas punishing cheaters that do not produce public goods. This lure-to-kill mechanism, mediated by a bifunctional T6SS effector, may offer new insights into the molecular mechanisms that maintain stability in microbial communities.

OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress.

The formation of reactive oxygen species is harmful and can destroy intracellular macromolecules such as lipids, proteins, and DNA, even leading to bacterial death. To cope with this situation, microbes have evolved a variety of sophisticated mechanisms, including antioxidant enzymes, siderophores, and the type VI secretion system (T6SS). However, the mechanism of oxidative stress resistance in Cupriavidus pinatubonensis is unclear. In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region. The study revealed that OxyR-regulated T6SS1 combats oxidative stress by importing iron into bacterial cells. Moreover, the T6SS1-mediated outer membrane vesicles-dependent iron acquisition pathway played a crucial role in the oxidative stress resistance process. Finally, our study demonstrated that the T6SS1 and siderophore systems in C. pinatubonensis exhibit different responses in combating oxidative stress under low-iron conditions, providing a comprehensive understanding of how bacterial iron acquisition systems function in diverse conditions.IMPORTANCEThe ability to eliminate reactive oxygen species is crucial for bacterial survival. Continuous formation of hydroperoxides can damage metalloenzymes, disrupt DNA integrity, and even result in cell death. While various mechanisms have been identified in other bacterial species to combat oxidative stress, the specific mechanism of oxidative stress resistance in C. pinatubonensis remains unclear. The importance of this study is that we elucidate the mechanism that OxyR-regulated T6SS1 combats oxidative stress by importing iron with the help of bacterial outer membrane vesicle. Moreover, the study highlights the contrasting responses of T6SS1- and siderophore-mediated iron acquisition systems to oxidative stress. This study provides a comprehensive understanding of bacterial iron acquisition and its role in oxidative stress resistance in C. pinatubonensis under low-iron conditions.

Salmonella T3SS-elicited inflammatory innate immune response inhibits type I IFN response in macrophages.

As a gram-negative intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium) invades different cell types including macrophages. Its infection in macrophages induces robust innate immune responses that are featured by proinflammatory and type I interferon (IFN) responses. The type III secretion systems (T3SSs) of S. Typhimurium play a crucial role in activating host inflammasome pathways. It has been recognized that the inflammasome pathways inhibit the type I IFN cascade. However, the potential role of T3SS in regulating the type I IFN response and the underlying mechanisms are largely unknown. In this study, we showed that S. Typhimurium infection activated strong proinflammatory, type I IFN and IFN-stimulated genes (ISGs) expression in macrophages. Furthermore, we showed that T3SS-defective S. Typhimurium mutant ΔinvC elicited attenuated inflammatory response but enhanced type I IFN and ISGs expression. Additionally, the inhibition of caspase-1 by a specific inhibitor VX-765 resulted in increased type I IFN response. Moreover, cell-permeable pan-caspase inhibitor Z-VAD-FMK also enhanced the type I IFN response upon S. Typhimurium infection. Intriguingly, compared with exponential phase S. Typhimurium infection, stationary phase bacteria triggered higher levels of type I IFN responses. Finally, the inhibition of caspase-1 by VX-765 substantially increased the intracellular S. Typhimurium burden. In conclusion, we demonstrated that the proinflammatory response induced by S. Typhimurium T3SS can inhibit the type I IFN response, which provides insight into the role of T3SS in orchestrating innate immunity during S. Typhimurium infection.

c-di-GMP inhibits the DNA binding activity of H-NS in Salmonella.

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria.

A Fur-regulated type VI secretion system contributes to oxidative stress resistance and virulence in Yersinia pseudotuberculosis.

The type VI secretion system (T6SS) is a widespread protein secretion apparatus deployed by many Gram-negative bacterial species to interact with competitor bacteria, host organisms, and the environment. Yersinia pseudotuberculosis T6SS4 was recently reported to be involved in manganese acquisition; however, the underlying regulatory mechanism still remains unclear. In this study, we discovered that T6SS4 is regulated by ferric uptake regulator (Fur) in response to manganese ions (Mn2+), and this negative regulation of Fur was proceeded by specifically recognizing the promoter region of T6SS4 in Y. pseudotuberculosis. Furthermore, T6SS4 is induced by low Mn2+ and oxidative stress conditions via Fur, acting as a Mn2+-responsive transcriptional regulator to maintain intracellular manganese homeostasis, which plays important role in the transport of Mn2+ for survival under oxidative stress. Our results provide evidence that T6SS4 can enhance the oxidative stress resistance and virulence for Y. pseudotuberculosis. This study provides new insights into the regulation of T6SS4 via the Mn2+-dependent transcriptional regulator Fur, and expands our knowledge of the regulatory mechanisms and functions of T6SS from Y. pseudotuberculosis.

Collaborative impact of bacterial exometabolites governing root microbiota formation.

The majority of the root microbiota formation derives from soil-dwelling microorganisms. The limited extent of thorough investigation leads to a dearth of knowledge concerning the intricate mechanisms of microbe-microbe interaction implicated in the establishment of root microbiota. Therefore, the taxonomic signatures in bacterial inhibition profiles were determined by in vitro testing of 39,204 binary interbacterial interactions. However, findings from genetic and metabolomic studies elucidated that co-functioning of the antimicrobial 2,4-d iacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites has significantly contributed to the potent inhibitory activities of the highly antagonistic Pseudomonas brassicacearum R401. Microbiota restoration with a core of Arabidopsis thaliana root commensals showed that these exometabolites possess a root niche-specific function in establishing root competence and inducing anticipated changes in root surroundings. Both biosynthetic operons are abundant in roots in natural habitats, indicating that these exometabolites co-functioning is an adaptive feature that helps Pseudomonad dominate the root microbiota.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

A Yersinia T6SS Effector YezP Engages the Hemin Uptake Receptor HmuR and ZnuABC for Zn2+ Acquisition.

Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn2+ acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn2+ into the periplasm by the YezP-Zn2+ complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn2+ from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. IMPORTANCE Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn2+ acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn2+ into the cytoplasm via the YezP-Zn2+ complex, and elucidation of the complete Zn2+ acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.

Isolation and Characterization of Three Pseudomonas aeruginosa Viruses with Therapeutic Potential.

As one of the most common pathogens of opportunistic and hospital-acquired infections, Pseudomonas aeruginosa is associated with resistance to diverse antibiotics, which represents a significant challenge to current treatment modalities. Phage therapy is considered a promising alternative to conventional antimicrobials. The characterization and isolation of new bacteriophages and the concurrent evaluation of their therapeutic potential are fundamental for phage therapy. In this study, we employed an enrichment method and a double-layer agar overlay to isolate bacteriophages that infect P. aeruginosa strains PAO1 and PA14. Three phages (named PA_LZ01, PA_LZ02, and PA_LZ03) were isolated and showed icosahedral heads and contractile tails. Following full-genome sequencing, we found that phage PA_LZ01 contained a genome of 65,367 bp in size and harbored 90 predicted open reading frames (ORFs), phage PA_LZ02 contained a genome of 57,243 bp in size and harbored 75 predicted ORFs, and phage PA_LZ03 contained a genome of 57,367 bp in size and carried 77 predicted ORFs. Further comparative analysis showed that phage PA_LZ01 belonged to the genus Pbunavirus genus, phage PA_LZ02 belonged to the genus Pamexvirus, and phage PA_LZ03 belonged to the family Mesyanzhinovviridae. Next, we demonstrated that these phages were rather stable at different temperatures and pHs. One-step growth curves showed that the burst size of PA_LZ01 was 15 PFU/infected cell, and that of PA_LZ02 was 50 PFU/infected cell, while the titer of PA_LZ03 was not elevated. Similarly, the biofilm clearance capacities of PA_LZ01 and PA_LZ02 were also higher than that of PA_LZ03. Therapeutically, PA_LZ01 and PA_LZ02 treatment led to decreased bacterial loads and inflammatory responses in a mouse model. In conclusion, we isolated three phages that can infect P. aeruginosa, which were stable in different environments and could reduce bacterial biofilms, suggesting their potential as promising candidates to treat P. aeruginosa infections. IMPORTANCE Phage therapy is a promising therapeutic option for treating bacterial infections that do not respond to common antimicrobial treatments. Biofilm-mediated infections are particularly difficult to treat with traditional antibiotics, and the emergence of antibiotic-resistant strains has further complicated the situation. Pseudomonas aeruginosa is a bacterial pathogen that causes chronic infections and is highly resistant to many antibiotics. The library of phages that target P. aeruginosa is expanding, and the isolation of new bacteriophages is constantly required. In this study, three bacteriophages that could infect P. aeruginosa were isolated, and their biological characteristics were investigated. In particular, the isolated phages are capable of reducing biofilms formed by P. aeruginosa. Further analysis indicates that treatment with PA_LZ01 and PA_LZ02 phages reduces bacterial loads and inflammatory responses in vivo. This study isolated and characterized bacteriophages that could infect P. aeruginosa, which offers a resource for phage therapy.

Metagenomics insights into responses of rhizobacteria and their alleviation role in licorice allelopathy.

BACKGROUND: Allelopathy is closely associated with rhizosphere biological processes, and rhizosphere microbial communities are essential for plant development. However, our understanding of rhizobacterial communities under influence of allelochemicals in licorice remains limited. In the present study, the responses and effects of rhizobacterial communities on licorice allelopathy were investigated using a combination of multi-omics sequencing and pot experiments, under allelochemical addition and rhizobacterial inoculation treatments. RESULTS: Here, we demonstrated that exogenous glycyrrhizin inhibits licorice development, and reshapes and enriches specific rhizobacteria and corresponding functions related to glycyrrhizin degradation. Moreover, the Novosphingobium genus accounted for a relatively high proportion of the enriched taxa and appeared in metagenomic assembly genomes. We further characterized the different capacities of single and synthetic inoculants to degrade glycyrrhizin and elucidated their distinct potency for alleviating licorice allelopathy. Notably, the single replenished N (Novosphingobium resinovorum) inoculant had the greatest allelopathy alleviation effects in licorice seedlings. CONCLUSIONS: Altogether, the findings highlight that exogenous glycyrrhizin simulates the allelopathic autotoxicity effects of licorice, and indigenous single rhizobacteria had greater effects than synthetic inoculants in protecting licorice growth from allelopathy. The results of the present study enhance our understanding of rhizobacterial community dynamics during licorice allelopathy, with potential implications for resolving continuous cropping obstacle in medicinal plant agriculture using rhizobacterial biofertilizers. Video Abstract.

Rethreading Design of Ratiometric roGFP2 Mimetic Peptide for Hydrogen Peroxide Sensing.

Reconstruction of the miniaturized peptide to mimic the tailored functions of protein has been attractive but challenging. Herein, initialized from the crystal structure of redox-sensitive green fluorescent protein-2 (roGFP2), we propose a practical approach to construct the roGFP2 mimetic peptide by rethreading the aromatic residues adjacent to the chromophore fragment. By fine-tuning the residues of peptides, a mini tetrapeptide (Cys-Phe-Phe-His) was designed, which can act as a hydrogen peroxide sensor using its ratiometric fluorescence. The roGFP2 mimetic tetrapeptide is biocompatible and photostable and has competitive fluorescent properties with roGFP2 by the virtue of its assembly induced emissions. We expand the ratiometric tetrapeptide for sensing hydrogen peroxide in acidic chambers. The results provide a promising approach for the artificial design of miniaturized peptides with the desired function.

Quantitative Ratiometric Biosensors Based on Fluorescent Ferrocene-Modified Histidine Dipeptide Nanoassemblies.

Fluorescent proteins (FPs) provide a ratiometric readout for quantitative assessment of the destination of internalized biomolecules. FP-inspired peptide nanostructures that can compete with FPs in their capacity are the most preferred building blocks for the synthesis of fluorescent soft matter. However, realizing a ratiometric emission from a single peptide fluorophore remains exclusive since multicolor emission is a rare property in peptide nanostructures. Here, we describe a bioinspired peptidyl platform for ratiometric intracellular quantitation by employing a single ferrocene-modified histidine dipeptide. The intensiometric ratio of green to blue fluorescence correlates linearly with the concentration of the peptide by three orders of magnitude. The ratiometric fluorescence of the peptide is an assembly-induced emission originating from hydrogen bonds and aromatic interactions. Additionally, modular design enables ferrocene-modified histidine dipeptides to use as a general platform for the construction of intricate peptides that retain the ratiometric fluorescent properties. The ratiometric peptide technique promises flexibility in the design of a wide spectrum of stoichiometric biosensors for quantitatively understanding the trafficking and subcellular fate of biomolecules.