Genomic and metabolomic insights into the antimicrobial compounds and plant growth-promoting potential of Bacillus velezensis Q-426.

BACKGROUND: The Q-426 strain isolated from compost samples has excellent antifungal activities against a variety of plant pathogens. However, the complete genome of Q-426 is still unclear, which limits the potential application of Q-426. RESULTS: Genome sequencing revealed that Q-426 contains a single circular chromosome 4,086,827 bp in length, with 4691 coding sequences and an average GC content of 46.3%. The Q-426 strain has a high degree of collinearity with B. velezensis FZB42, B. velezensis SQR9, and B. amyloliquefaciens DSM7, and the strain was reidentified as B. velezensis Q-426 based on the homology analysis results. Many genes in the Q-426 genome have plant growth-promoting activity, including the secondary metabolites of lipopeptides. Genome mining revealed 14 clusters and 732 genes encoding secondary metabolites with predicted functions, including the surfactin, iturin, and fengycin families. In addition, twelve lipopeptides (surfactin, iturin and fengycin) were successfully detected from the fermentation broth of B. velezensis Q-426 by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS), which is consistent with the genome analysis results. We found that Q-426 produced indole-3-acetic acid (IAA) at 1.56 mg/l on the third day of incubation, which might promote the growth of plants. Moreover, we identified eighteen volatile compounds (VOCs, including 2-heptanone, 6-methylheptan-2-one, 5-methylheptan-2-one, 2-nonanone, 2-decanone, 2-undecanone, 2-dodecanone, 2-tridecanone, 2-tetradecanone, 2-nonadecanone, pentadecanoic acid, oleic acid, dethyl phthalate, dibutyl phthalate, methyl (9E,12E)-octadeca-9,12-dienoate), pentadecane, (6E,10E)-1,2,3,4,4a,5,8,9,12,12a-decahydro-1,4-methanobenzo[10]annulene, and nonanal) based on gas chromatograph-mass spectrometer (GC/MS) results. CONCLUSIONS: We mined secondary metabolite-related genes from the genome based on whole-genome sequence results. Our study laid the theoretical foundation for the development of secondary metabolites and the application of B. velezensis Q-426. Our findings provide insights into the genetic characteristics responsible for the bioactivities and potential application of B. velezensis Q-426 as a plant growth-promoting strain in ecological agriculture.

The effects of L-arginine on protein stability and DNA binding ability of SaeR, a transcription factor in Staphylococcus aureus.

The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production and stability of the recombinant protein SaeR, l-arginine (L-Arg) was added to the purification buffers. L-Arg enhanced the solubility and stability of the recombinant protein SaeR. The thermal denaturation temperature of SaeR in 10 mM L-Arg buffer was significantly increased compared to the buffer without L-Arg. Microscale Thermophoresis (MST) analysis results showed that the SaeR protein could bind to the P1 promoter under both phosphorylated and non-phosphorylated status in buffer containing 10 mM L-Arg. These results illustrate an effective method to purify SaeR and other proteins.

Developing nucleoside tailoring strategies against SARS-CoV-2 via ribonuclease targeting chimera.

In response to the urgent need for potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics, this study introduces an innovative nucleoside tailoring strategy leveraging ribonuclease targeting chimeras. By seamlessly integrating ribonuclease L recruiters into nucleosides, we address RNA recognition challenges and effectively inhibit severe acute respiratory syndrome coronavirus 2 replication in human cells. Notably, nucleosides tailored at the ribose 2'-position outperform those modified at the nucleobase. Our in vivo validation using hamster models further bolsters the promise of this nucleoside tailoring approach, positioning it as a valuable asset in the development of innovative antiviral drugs.

Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.

Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.

[Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum].

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 ß-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Quantitative Proteomic Analysis of Outer Membrane Vesicles from Fusobacterium nucleatum Cultivated in the Mimic Cancer Environment.

Fusobacterium nucleatum is a Gram-negative bacterium that has been identified as an important pathogenic gut bacterium associated with colorectal cancer. Compared with the normal intestine, the pH value of the tumor microenvironment is weakly acidic. The metabolic changes of F. nucleatum in the tumor microenvironment, especially the protein composition of its outer membrane vesicles, remain unclear. Here, we systematically analyzed the effect of environmental pH on the proteome of outer membrane vesicles (OMVs) from F. nucleatum by tandem mass tag (TMT) labeling-high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 991 proteins were identified in acidic OMVs (aOMVs) and neutral OMVs (nOMVs), including known virulence proteins and putative virulence proteins. Finally, 306 upregulated proteins and 360 downregulated proteins were detected in aOMVs, and approximately 70% of the expression of OMV proteins was altered under acidic conditions. A total of 29 autotransporters were identified in F. nucleatum OMVs, and 13 autotransporters were upregulated in aOMVs. Interestingly, three upregulated autotransporters (D5REI9, D5RD69, and D5RBW2) show homology to the known virulence factor Fap2, suggesting that they may be involved in various pathogenic pathways such as the pathway for binding with colorectal cancer cells. Moreover, we found that more than 70% of MORN2 domain-containing proteins may have toxic effects on host cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that a number of proteins were significantly enriched in multiple pathways involving fatty acid synthesis and butyrate synthesis. Seven metabolic enzymes involved in fatty acid metabolism pathways were identified in the proteomic data, of which 5 were upregulated and 2 were downregulated in aOMVs, while 14 metabolic enzymes involved in the butyric acid metabolic pathway were downregulated in aOMVs. In conclusion, we found a key difference in virulence proteins and pathways in the outer membrane vesicles of F. nucleatum between the tumor microenvironment pH and normal intestinal pH, which provides new clues for the prevention and treatment of colorectal cancer. IMPORTANCE F. nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. OMVs have been demonstrated to play key roles in pathogenesis by delivering toxins and other virulence factors to host cells. By employing quantitative proteomic analysis, we found that the pH conditions could affect the protein expression of the outer membrane vesicles of F. nucleatum. Under acidic conditions, approximately 70% of the expression of proteins in OMVs was altered. Several virulence factors, such as type 5a secreted autotransporter (T5aSSs) and membrane occupation and recognition nexus (MORN) domain-containing proteins, were upregulated under acidic conditions. A large number of proteins showed significant enrichments in multiple pathways involving fatty acid synthesis and butyrate synthesis. Proteomics analysis of the outer membrane vesicles secreted by pathogenic bacteria in the acidic tumor microenvironment is of great significance for elucidating the pathogenicity mechanism and its application in vaccine and drug delivery vehicles.

Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum.

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Until now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here, we report a novel, recombinant, two-component, response regulator protein ArlR from the genome of F. nucleatum strain ATCC 25,586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein with the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 CodonPlus (DE3) RIL that was later induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG) for 8 h at 25 °C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3 kDa with approximately 90% purity after gel filtration chromatography. Because ArlR was biologically active after its purification, it accepted the corresponding phosphorylated histidine kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase was about 2.1 µM, whereas the binding constant between ArlR and its operon was 6.4 µM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

MeWRKY IIas, Subfamily Genes of WRKY Transcription Factors From Cassava, Play an Important Role in Disease Resistance.

Cassava (Manihot esculenta Crantz) is an important tropical crop for food, fodder, and energy. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) occurs in all cassava growing regions and threatens global cassava production. WRKY transcription factor family plays the essential roles during plant growth, development, and abiotic or biotic stress. Particularly, previous studies have revealed the important role of the group IIa WRKY genes in plant disease resistance. However, a comprehensive analysis of group IIa subfamily in cassava is still missing. Here, we identified 102 WRKY members, which were classified into three groups, I, II, and III. Transient expression showed that six MeWRKY IIas were localized in the nucleus. MeWRKY IIas transcripts accumulated significantly in response to SA, JA, and Xam. Overexpression of MeWRKY27 and MeWRKY33 in Arabidopsis enhanced its resistance to Pst DC3000. In contrast, silencing of MeWRKY27 and MeWRKY33 in cassava enhanced its susceptibility to Xam. Co-expression network analysis showed that different downstream genes are regulated by different MeWRKY IIa members. The functional analysis of downstream genes will provide clues for clarifying molecular mechanism of cassava disease resistance. Collectively, our results suggest that MeWRKY IIas are regulated by SA, JA signaling, and coordinate response to Xam infection.

Insights into the structure and function of the histidine kinase ComP from Bacillus amyloliquefaciens based on molecular modeling.

The ComPA two-component signal transduction system (TCS) is essential in Bacillus spp. However, the molecular mechanism of the histidine kinase ComP remains unclear. Here, we predicted the structure of ComP from Bacillus amyloliquefaciens Q-426 (BaComP) using an artificial intelligence approach, analyzed the structural characteristics based on the molecular docking results and compared homologous proteins, and then investigated the biochemical properties of BaComP. We obtained a truncated ComPS protein with high purity and correct folding in solution based on the predicted structures. The expression and purification of BaComP proteins suggested that the subdomains in the cytoplasmic region influenced the expression and stability of the recombinant proteins. ComPS is a bifunctional enzyme that exhibits the activity of both histidine kinase and phosphotransferase. We found that His571 played an obligatory role in the autophosphorylation of BaComP based on the analysis of the structures and mutagenesis studies. The molecular docking results suggested that the HATPase_c domain contained an ATP-binding pocket, and the ATP molecule was coordinated by eight conserved residues from the N, G1, and G2 boxes. Our study provides novel insight into the histidine kinase BaComP and its homologous proteins.

Genome-wide identification of cassava MeRboh genes and functional analysis in Arabidopsis.

Plant respiratory burst oxidase homolog (Rboh) gene family encodes NADPH oxidases, and plays important roles in the production of reactive oxygen species (ROS), plant signaling, growth and stress responses. Cassava is an important starchy crops in tropical region. Environmental stresses, such as drought, pathogen, have caused great yield loss. The mechanisms of stress response are little known in MeRBOH family of cassava. Investigation of Rboh genes response to disease may provide a clue for clarification the disease resistance mechanisms. In this study, eight MeRboh genes were identified from the cassava genome. Comparisons of gene structure, protein motifs, and a phylogenetic tree showed conservation of Rboh gene families in cassava, Arabidopsis and rice. Transcript levels of most MeRboh genes increased following treatment with a pathogen, Xanthomonas axonopodis pv. manihotis, or with phytohormones salicylic acid or jasmonic acid. Analysis of cis-acting elements also indicated that MeRboh genes could response to light, hormone, abiotic and biotic stress. Prediction of miRNA target and post-translation modification sites of MeRboh suggested possible regulations of miRNA and protein phosphorylation; and transient expression of MeRboh in cassava protoplasts confirmed their localization on plasma membrane. Expression of MeRbohB, MeRbohF partially complemented PAMP responses in Arabidopsis rboh mutants, including the expression of PTI marker FRK1, ROS production, peroxide accumulation and callose deposition. It suggesting that MeRbohB and MeRbohF may participate in the PTI pathway and contributed to ROS production triggered by pathogens. Moreover, overexpression of MeRbohB and MeRbohF enhanced the resistance of Arabidopsis against Pseudomonas syringae pv. tomato DC3000. Together, these results suggest the evolutionary conservation of MeRboh gene family and their important role in the immune response and in regulating the plant disease resistance, providing a foundation for revealing molecular mechanisms of cassava disease resistance.