Streptomyces can produce various active secondary metabolites, which can be widely used in medical, industrial, agricultural, and other fields. The secondary metabolite synthesis in Streptomyces is regulated by pathway-specific, pleiotropic, and global regulatory genes. The two-component system, as the main signal transduction system in prokaryotes, participates in various physiological and biochemical reactions of Streptomyces and can globally regulate secondary metabolites. The deletion or overexpression of specific two-component system genes can significantly affect the biosynthesis of secondary metabolites. Identifying the functions of two-component systems and elucidating their regulatory mechanisms can contribute to enhancing the production efficiency of secondary metabolites by genetic engineering. This paper reviews the research trends of two-component systems in various Streptomyces species such as Streptomyces albidoflavus in recent years and particularly summarizes and elaborates on the regulatory mechanisms of their secondary metabolite synthesis.

[Objective] Transcription is the first step in gene expression, and the mRNA abundance to a certain extent determines the final protein expression abundance. Recent studies have found that different ribosome-binding sites (RBSs) located in the 5′ untranslated region (5′-UTR) can affect the mRNA abundance of the downstream gene. From the perspective of regulatory factors in the mRNA degradation process, the effect may be attributed to the binding strength between the Shine-Dalgarno (SD) sequence and the ribosome and the local secondary structure of the 5′-UTR. [Methods] We constructed a 5′-UTR mutant library with a size of 528. High-throughput sequencing was employed to efficiently collect the information on the mRNA abundance of downstream egfp corresponding to various 5′-UTR variants. The effectiveness was verified by RT-qPCR. [Results] The association between abundance of each mRNA mutant and its corresponding 5′-UTR sequence was analyzed. The results showed that the SD sequence with moderate to strong binding strength to the ribosome was most conducive to maintaining high mRNA abundance. Too high or low binding strength will lead to a reduction in the mRNA abundance. The completely conserved core SD sequence (GGAGG) was the key to ensuring high binding strength, and the decline in conservation would cause a significant decrease in the mRNA abundance. When the SD sequence was similar among different 5′-UTR variants, i.e.,the binding strength of the SD sequence to the ribosome was comparable, the local secondary structure of the 5′-UTR was instable and the abundance of corresponding mRNA was high. [Conclusion] This study delves into the regulatory effects of 5′-UTR sequence features 5′-UTR on the mRNA abundance and establishes a qualitative model of their interrelationships, providing a reference for the rational design of regulatory elements in metabolic engineering and gene circuits.

Saline-alkali stress is one of the main abiotic constraints limiting plant growth and development. Endophytic bacteria can enhance the stress tolerance of host plants by increasing osmotic adjustment substances and boosting antioxidant enzyme activities. [Objective] To isolate and identify saline-alkali tolerant endophytic bacteria from the roots of alfalfa grown in saline-alkali soil and evaluate them regarding the saline-alkali tolerance, plant growth-promoting traits, effects on alfalfa growth under saline-alkali stress, and colonization. [Methods] Saline-alkali tolerant endophytes were isolated by the tissue homogenization method from alfalfa roots. Strains were identified by morphological observation, 16S rRNA gene-based phylogenetic analysis, and physiological and biochemical assays. Multiple plant growth-promoting traits were assayed in vitro. A greenhouse pot experiment was conducted to assess the effect of the selected strain on alfalfa growth under saline-alkali conditions. Colonization of strain Z-1 in alfalfa roots was visualized by green fluorescent protein tagging and laser scanning confocal microscopy. [Results] Pseudomonas moraviensis Z-1 was successfully isolated from the roots of alfalfa growing in saline-alkali soil. The endophytic bacterial strain tolerated 4% NaCl and pH 9.0 and displayed the ability to produce 1-aminocyclopropane-l-carboxylate deaminase, siderophores, indole-3-acetic acid, and soluble phosphorus. Under saline-alkali conditions, inoculation with Z-1 significantly increased the dry weights of the aboveground parts, root vigor, and soluble protein content of alfalfa. Moreover, the strain significantly increased catalase, peroxidase, and superoxide dismutase activities and decreased the hydrogen peroxide, superoxide anion, and malondialdehyde content (P<0.05). Confocal microscopy confirmed successful colonization of Z-1 in alfalfa roots at 7.57×10⁴ CFU/g. [Conclusion] The saline-alkali tolerant endophytic bacterium Z-1 plays a vital role in promoting alfalfa growth and enhancing its tolerance to saline-alkali stress. It represents a promising candidate for developing microbial preparations to ameliorate saline-alkali soil.

[Objective] To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus (IBV) as a vaccine to provide comprehensive protection. Additionally, it explores the potential of polyclonal yolk antibodies (IgY) harvested from laying hens immunized with the rMKIBV vaccine in the prevention and control of IBV. [Methods] The antigenic epitope sequences of IBV, obtained from online databases, were compared with sequences of representative IBV strains from GenBank. Flexible peptides were designed to link all antigenic peptides. The constructed amino acid sequence was analyzed, reverse-translated, codon-optimized, and then inserted into the pET-28a(+) cloning vector. The recombinant vector was introduced into Escherichia coli for expression. The purified, desalted, and endotoxin-removed rMKIBV protein was used as a vaccine to immunize animals for investigation of its immunogenicity and ability to stimulate specific IgY production in laying hens. [Results] The retrieved IBV antigenic epitope sequences showed high similarity with the published N and S protein sequences of 22 representative IBV strains. The predicted isoelectric point and molecular weight of rMKIBV were 10.25 and 63.39 kDa, respectively. The secondary structure of rMKIBV included a high proportion of random coils, which suggested strong antigenicity. High-purity rMKIBV was obtained from E. coli transformed with the recombinant plasmid pET-28a-mkibv. This protein specifically bound to anti-His-tag antibodies, N protein antibodies, and S protein antibodies. The mice immunized with this protein showed increases in the spleen index (P<0.05), elevations in the levels of serum-specific IgG antibodies (P<0.01) and IFN-γ (P<0.05), and no significant change in the IL-2 level. Immunized laying hens successfully produced IgY in egg yolks, with specific IgY antibody levels significantly increasing. Moreover, the IgY antibody titer gradually rose after immunization, reaching the peak after about 50 days and then gradually declining to reach a stable level. [Conclusion] We successfully constructed and expressed the recombinant protein rMKIBV. The protein demonstrated good immunogenicity, stimulating specific antibody production in both mice and laying hens. Notably, the IgY extracted from the yolks of immunized laying hens offers a novel approach to IBV prevention and control. These findings hold significant scientific and practical value for the development of vaccines against IBV.

[Objective] To screening stress-tolerant and high-yielding rhizobia with growth-promoting effects on Sesbaniacannabina and provide rhizobia resources for efficient cultivation of S. cannabina in saline-alkali soil. [Methods] The culture method was used to isolate endophytic rhizobia from S. cannabina ‘Zhongkejing 1’. Based on 16S rRNA gene and whole genome sequencing, the strains were identified, and their stress tolerance and plant growth-promoting characteristics were evaluated. Their growth-promoting effects on the original host variety and other materials of S. cannabina were verified. [Results] The rhizobia isolated from the root nodule samples of S. cannabina ‘Zhongkejing 1’ were identified as a species belonging to Rhizobium. Based on the ANI and dDDH values of the whole genome sequence, the strain was identified as a new species of Rhizobium and named Rhizobium sesbaniae ZK1^T. R. sesbaniae ZK1^T can tolerate a NaCl concentration of 2.0% and survive within the range of pH 4.0-10.0, and it had the ability to dissolve organophosphorus compounds. Pot experiments were conducted to evaluate the effects of R. sesbaniae ZK1^T on the growth and nodulation of different materials of S. cannabina. The results revealed that R. sesbaniae ZK1^T promoted the growth and nodulation of these materials, while it had a more efficient symbiotic relationship with the host variety. [Conclusion] The isolated new species R. sesbaniae ZK1^T plays a role in promoting the growth and nodulation of S. cannabina and can tolerate severe acid, alkali, and salt stress. The findings have important theoretical significance and a practical value for the efficient improvement of plant-microorganism interactions in marginal land.

One Health integrates the health of the environment, animals, and humans, involving food safety, environmental hygiene, and animal and human health. Currently, antibiotic resistance is exacerbating worldwide, seriously hindering the achievement of One Health. Phages, as viruses with a century-long application history and the ability to specifically kill bacteria, bring new hope for addressing antibiotic-resistant bacterial infections. This article reviews the development history, diversity, and applications of phages in food safety and the environment, animals, and humans, with the aim of providing references for the application of phages in the era of One Health.

Phosphorus is an essential nutrient element for plant growth and development. However, the available phosphorus in soil is extremely limited due to the presence of large amounts of organic phosphorus that is difficult to be degraded, with phytate (inositol hexaphosphate) accounting for a significant proportion. Phytases can efficiently hydrolyze phytate and release available phosphorus. [Objective] By taking advantage of the efficient hydrolysis ability of phytase, the phytase gene was gene modified in the indigenous bacteria of black soil to increase the available phosphorus content in the soil. [Methods] We employed the anchored protein pGSA for surface display of the bacterial phytaseAppA, thereby enhancing the stability and enzymatic activity of the protein as well as improving the substrate contact efficiency. Furthermore, leveraging the CRISPR-targeted gene editing technology, we precisely integrated the surface-displayed phytase fusion protein into the 16S rRNA gene of Ralstonia pickettii G3 genome, isolated from black soil to overcome the dependence of protein expression on vectors. [Results] The 16S rRNA gene site could be used as a target for gene modification without significant effect on the proliferation of the bacteria. The phytase-modified engineered bacteria showed a more than 8-fold increase in the hydrolytic ability of phytate and functioned in a wide pH range. After this indigenous engineered bacterial strain was applied to black soil, the soil phytase activity significantly increased, and the available phosphorus content rose by nearly 30%. [Conclusion] Modifying phytase by gene editing can promote the hydrolysis of phytate, increase the content of available phosphorus, and improve the phosphorus availability in the soil.

Yeast β-glucan has been widely used as a dietary supplement for its multiple biological activities, including immune stimulation. Previous studies in our laboratory have shown that yeast chs3Δ spores exposed in the glucan layer can activate immune effects more effectively than trophoblasts. However, chs3Δ spores are formed within the ascospores and are encapsulated by the ascospore cell wall, which greatly limits their direct application. [Objective] To explore whether the lysates of chs3Δ asci have the potential to be used as novel immune-stimulating dietary supplements. [Methods] We prepared chs3Δ asci lysates by ultrasonic freeze drying, enzymatic freeze drying, and enzymatic-assisted ultrasonic freeze drying and then explored their immune effects in depth. [Results] The chs3Δ asci lysate prepared by ultrasonic freeze drying induced the highest level of inflammatory cytokines compared with the vegetative cell lysate and chs3Δ spores, and the asci lysate achieved immunostimulatory responses through activation of Dectin-1 receptor. Further studies showed that the chs3Δ asci lysate also exhibited immunostimulatory activity and trained immunity-inducing ability in mice. [Conclusion] The chs3Δ asci lysate has great potential as a highly effective immunostimulatory dietary supplement.

Nitrogen fertilizer is an important chemical fertilizer for agricultural planting and an important fertility factor for increasing crop yields. Lack or excess of nitrogen fertilizer in soil will lead to soil acidification, soil consolidation, low crop yields and so on. Nitrogen-fixing bacteria can reduce nitrogen in the air to ammonia that is beneficial to crops through the action of nitrogenase. This process helps improve soil quality and subsequently promote crop growth. [Objective] To obtain nitrogen-fixing bacteria from the black soil of northeast China and explore the effects of nitrogen-fixing bacteria on soil quality and maize growth, thus providing excellent strain sources for the development of microbial agents suitable for the environment of black soil in northeast China. [Methods] We employed microbial isolation, culture, and functional characterization to measure the nitrogen fixation, phosphorus solubilization, and indole-3-acetic acid (IAA) secretion of the screened nitrogen-fixing bacteria. Pot experiments and soil physical and chemical tests were carried out to evaluate the effects of nitrogen-fixing bacteria on soil quality and maize growth. [Results] Three strains of nitrogen-fixing bacteria were obtained from the black soil of northeast China. Among them, Paenibacillus sp. AHC-20 had higher nitrogen-fixing ability, while Raoultella sp. Z93 and Paraburkholderia sp. W22 were multifunctional strains capable of fixing nitrogen, solubilizing phosphorus, and producing IAA at the same time. The three nitrogen-fixing strains were then applied to the black soil planted with maize. The plant height, biomass, and chlorophyll content of maize significantly increased in the chemical fertilizer reduction+AHC-20 group compared with those in the control group with application of only chemical fertilizer. In addition, the content of inorganic carbon, organic carbon, organic matter, ammonium nitrogen, and nitrate nitrogen in black soil also increased significantly, which indicated that the efficient nitrogen-fixing bacterial strain AHC-20 promoted maize growth upon chemical fertilizer reduction and improved the fertility of black soil. [Conclusion] Nitrogen-fixing bacteria in black soil can effectively promote the growth of maize and improve the quality of black soil to achieve fertilizer reduction without compromising crop yields, showing the potential for the development of nitrogen-fixing microbial agents.