[Objective] To investigate the regulatory role of the transcription factor BldM in the morphological development and antibiotic synthesis ofStreptomyces pactum Act12, a biocontrol strain with multiple effects.[Methods] ThebldM-deleted mutant strain ∆bldM and thebldM-overexpressing mutant strain OE-bldM were constructed by genetic engineering. The scanning electron microscopy, antibacterial experiment, high performance liquid chromatography, and real-time quantitative PCR were employed to compare the morphological development, growth rate, oligomycin yield, and resistance to pathogens, respectively, between ∆bldM, OE-bldM, and the wild-type strain Act12.[Results] The sequencing results proved that ∆bldM and OE-bldM were successfully constructed. ∆bldM showed significantly reduced production of oligomycin D and was incapable of forming aerial hyphae. OE-bldM presented dense aerial hyphae and active sporulation. Compared with the wild type, OE-bldM showed an increase of 23% in the yield of oligomycin D and the up-regulation of 2–3 times in the transcriptional levels of the genes encoding oligomycin core synthetase. Moreover, the antimicrobial activity of OE-bldM remarkably enhanced.[Conclusion] The global transcriptional regulator BldM can not only affect the formation of aerial hyphae and sporulation but also participate in the positive regulation of oligomycin synthesis in Act12. The results of this study supplement the knowledge about the regulatory function of BldM and provide a reference for further research on the growth, metabolism, and regulation mechanism ofS.pactum Act12.

[Objective] To explore the reasons for differences in the C-methylation programming of non-reducing polyketide synthases (NR-Pkss).[Methods] We used bioinformatics tools and AlphaFold 2 to compare the domain sequences and structures of the NR-Pkss involved in the synthesis ofMonascus pigment and citrinin inMonascus ruber M7, i.e., Mr-PksPT and Mr-PksCT. Furthermore, we employed molecular docking to compare the binding of C-methyltransferase domains (CMeTs) with other domains and the intermediates of the two NR-Pkss.[Results] The large differences of the overall structure and the high similarity of domain sequence and structure between the two NR-Pkss suggested that the differences of C-methylation programming between NR-Pkss may be resulted from domain interactions. The CMeT of Mr-PksCT was more likely to bind to the acyl carrier protein (ACP) carrying the substrate than that of Mr-PksPT, making the intermediate more easily catalyzed by CMeT. Moreover, CMeT had lower binding free energy to methyl receptor substrate than theβ-ketosynthase domain (KS).[Conclusion] The CMeTs of NR-Pkss can affect the C-methylation of the products by competing with KS. The findings provide a new idea for the study of C-methylation programming of Pkss.

[Objective] To screen out a fungal strain that can efficiently assimilate ammonia nitrogen, reveal the metabolome differences of the strain in different media and the changes in the amino acid content of the feed fermented with the strain, and clarify the mechanism of its ammonia assimilation.[Methods] Seven strains ofTrichoderma, 7 strains ofAspergillus niger, and 9 strains ofAspergillus oryzae were cultured in the media with (NH₄)₂SO₄ as the only nitrogen source. The strains with high ammonia nitrogen use efficiency and glutamine synthetase (GS) activity were selected for comparison of the metabolic differences in potato dextrose agar (PDA) plates and inorganic nitrogen plates by non-targeted metabonomics. Furthermore, the crude protein and organic nitrogen content in the feed fermented with different strains was determined, and the changes in amino acid content in the fermented feed extract were measured by amino acid-targeted metabolomics.[Results] The utilization rate of ammonia nitrogen and the glutamine synthetase activity ofA. oryzae MQ28 were 54.46% and 0.61 μmol/(h·g), respectively, which were higher than those of other strains (P < 0.05). The comparative metabonomics analysis suggested that MQ28 was associated with the metabolism of multiple amino acids during ammonia assimilation. MQ28 fermentation increased the crude protein and organic nitrogen in the feed by 22.25% and 35.83% (P < 0.05), respectively. Furthermore, MQ28 fermentation increased the total amino acid content in feed extract from 31.86 mmol/100 g to 57.69 mmol/100 g (P < 0.05). Specifically, it increased the content of 14 amino acids such as threonine, lysine, and arginine, glutamic acid (by 3.46 folds), and glutamine (by 99 folds) (P < 0.05).[Conclusion] To sum up,A.oryzae MQ28 has high ammonia nitrogen utilization capacity. It may regulate the ammonia assimilation process through the synthesis of glutamine to regulate amino acid metabolism and can be used as an elite strain for the production of single-cell protein.

[Objective] To reveal the mechanisms ofPseudomonas putida Y-9 in actively regulating the extracellular and intracellular pH homeostasis during ammonia oxidation.[Methods] Y-9 was cultured in the nitrification media with initial pH 7.19 and 9.40, respectively, for 48 h. Metabolomics was employed to compare the differential metabolites and predict dissociation constant (pKa) during the ammonia oxidation. Transcriptomics was employed to compare the genes regulating.[Results] In the medium with initial pH 7.19, Y-9 produced maltitol to raise extracellular pH, and up-regulated the expression of the genes related to deaminase, deiminase, and cation transport to maintain intracellular pH stability. In the medium with initial pH 9.40, Y-9 produced acidic substances such as 5-aminovaleric acid 3 and oxamic acid to lower extracellular pH and regulated the expression of the genes associated with NADH dehydrogenase, cytochromes, ATP synthase, and amino acid transport to maintain intracellular acidity.[Conclusion] This study revealed the novel phenomenon of Y-9's extracellular pH stabilizing capacity and investigated its intracellular pH homeostasis mechanism. The findings enrich our knowledge about microorganism-environment interactions, and provide a theoretical basis for further understanding the pH stabilization mechanism in microbial denitrification processes.

[Objective] Cyanophages, the viruses specifically infecting cyanobacteria, are ubiquitous in water environments. They play a role in regulating the population dynamics and density of cyanobacteria and promote the biogeochemical cycling of the aquatic ecosystem. This study aims to isolate and identify a cyanophage.[Methods] A novel cyanophage Yong-L2-223 was isolated from fresh water samples with marineSynechococcus sp. PCC 7002 as the indicator host. The host range, genome sequence, open reading frames (ORFs), and phylogenetic relationship of Yong-L2-223 were studied.[Results] The host range tests against 31 strains of cyanobacteria showed that Yong-L2-223 could infect the indicator host PCC 7002 (Synechococcales) and two freshwater strainsMicrocystis viridis FACHB-1342 (Chroococcales) andAphanizomenon flos-aquae FACHB-1209 (Nostocales) from the Dianchi Lake. The infection of the cyanobacterial strains from both the seawater and freshwater samples indicated that Yong-L2-223 was a euryhaline cyanophage. Yong-L2-223 was myovirus-like, consisting of an icosahedral head (about 60 nm in diameter) and a contractile tail (about 136 nm in length). The genome (double-stranded DNA) of Yong-L2-223 had a length of 65 725 bp, with the G+C content of 58.6% and 100 ORFs. It was predicted to carry theCas4 gene, gene transfer factor (GTA) gene, auxiliary metabolic genes (AMGs), and a gene cluster for the synthesis of pre-Q₀. These genes may contribute to the adaptation and infection of the cyanophage in cyanobacteria of three orders. The pairwise sequence comparison (PASC) illustrated that the highest similarity sharing by cyanophage Yong-L2-223 and all the viruses in the current genome databases was only 3.78%, far below the genus boundary cut-off of 70% defined by the International Committee on Taxonomy of Viruses. In the phylogenetic tree based on the whole proteomes, Yong-L2-223 formed an independent branch, with long evolutionary distances from other phages.[Conclusion] Yong-L2-223 is a new genus of theCaudoviricetes class. For the first time, we used a marine cyanobacterial strain as the indicator host to isolate and obtain a novel cyanophage from freshwater, which broadened the understanding of cyanophages, enriched cyanophage genome database, and laid a foundation for the development of cyanophage resources.

Gray mold caused byBotrytis cinerea is one of the major diseases affecting tomato production. The currently used fungicides are gradually restricted due to residues, pathogen resistance, and food safety. Therefore, screening out antagonistic microorganisms has gradually become an effective approach for the biocontrol of gray mold. [Objective] We screened out endophytic strains capable of endowing plants with disease resistance and promoting plant growth from tomato plants and evaluated their biocontrol potential, aiming to provide a theoretical basis for developing a new approach for the biocontrol of tomato gray mold.[Methods] The endophytic bacteria and fungi were isolated from different parts of tomato plants by the tissue culture method, and the candidate strains were preliminarily identified by 16S rRNA and ITS sequence analysis. The endophytes with antagonistic activity againstB.cinerea were screened by confrontation culture and fruit inoculationin vitro. Furthermore, we examined the abilities of the strain to secrete indole-3 acetic acid (IAA), protease, and siderophores and the promoting effects on the growth ofArabidopsis thaliana and tomato seedlings.[Results] A total of 72 endophytic bacterial strains and 31 endophytic fungal strains were isolated from different parts of tomato plants. An endophytic bacterium FQ-G3 with strong inhibitory activities against several pathogens was screened out and was identified asBacillus velezensis. FQ-G3 showed the inhibition rate of 80.93% againstB.cinereain vitro and inhibited the mycelial expansion on tomato fruitsin vivo. The strain could secrete IAA, protease, and siderophores, and promote the growth ofA.thaliana and tomato seedlings.[Conclusion] The endophytic strain FQ-G3 isolated from tomato plants endows plants with disease resistance and promotes plant growth, and thus can serve as a candidate for the prevention and control of gray mold. The findings enrich the tomato endophyte resources and provide support for the control of gray mold and the growth promotion of tomato.

[Objective] Bacillus velezensis SH-1471 (CCTCC No. M 2022923, Patent No. ZL 2022 1 1479280.X) is a strain that can control soil-borne diseases of crops and promote soil nutrient conversion and crop growth. This study aims to explore its potential biological activity and the optimum fermentation conditions, so as to promote the industrialization and commercial development of this strain.[Methods] A strain SH-1471 was identified based on the morphological, physiological, and biochemical characteristics and the 16S rRNA gene andgyrB-based phylogenetic trees. PCR was carried out to detect antibiotic synthesis genes in the strain. The inhibitory spectrum of the strain was measured by the plate confrontation assay and the fermentation liquid inhibition test. The abilities of the strain to produce enzymes, solubilize phosphorus and potassium, fix nitrogen, and secrete siderophoresin vitro were measured. The fermentation conditions were optimized by single factor tests and response surface methodology withOD₆₀₀ value and inhibition rate as indicators. The growth-promoting effect of the fermentation liquid on tomato plants and the control effect of the fermentation liquid on tomatoFusarium wilt before and after optimization were determined by indoor pot experiments.[Results] Strain SH-1471 was identified asB.velezensis, carrying the antibiotic synthesis genessrfA,fenB,ituA,ituD, andbymA. It had strong antagonistic effects on 8 pathogenic microorganisms such asFusarium oxysporum,Alternaria alternate, andExserohilum turcicum. Moreover, the strain was capable of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores. The optimal medium formula for the fermentation of SH-1471 was composed of 17.92 g/L sucrose, 16.95 g/L soybean powder, 2.88 g/L magnesium sulfate, and 5.0 g/L yeast extract. The optimum fermentation conditions were pH 7.5, 33 ℃, 220 r/min, and 20–24 h. After optimization, the inhibition rate,OD₆₀₀, and potted control efficiency of the fermentation liquid reached 94.08% (increasing by 15.6%), 3.28 (increasing by 36.7%), and 93.8%, respectively. Moreover, strain SH-1471 significantly improved plant height, stem circumference, root length, root weight, and fresh weight and dry weight of the aboveground part of tomato seedlings.[Conclusion] B.velezensis SH-1471 carries rich genes for antibiotic synthesis, has the abilities of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores, and demonstrates strong inhibitory effects on a variety of pathogens. It can significantly reduce the incidence of tomatoFusarium wilt and improve the agronomic traits of tomato seedlings. Therefore, the strain has broad application prospects in the biocontrol of plant diseases and the promotion of plant growth.

[Objective] This study aims to observe the changes of the fecal microbial communities in the dairy cows with subacute rumen acidosis (SARA) induced by a high-concentrate diet and modulated by the addition of vitamin E (VE). The potential effects on the metabolism of dairy cows were evaluated to provide data for exploring the physiological mechanisms of SARA.[Methods] Seven multiparous Holstein cows with rumen fistulas were selected for this trial which was carried out in three phases of 18 days each. The first phase was the control (CON) phase, with a concentrate-to-forage ratio of 50:50 in the diet (dry matter basis). The second phase was the induction (HG) phase, in which the forage was replaced with wheat flour at 15% of the diet (dry matter basis) to induce SARA. The third phase was the regulation (HGE) phase, in which VE was added at 12 000 IU/d/cow on the basis of the diet in the HG phase. The feces samples were collected on day 18 in each phase, and the microbial communities in the samples were examined.[Results] The fecal microbial community structure showed significant differences between three phases and the Shannon index in the HG phase was lower than that in the CON phase (P < 0.05). The HG phase had higher relative abundance ofProteobacteria andBlautia and lower relative abundance of unidentified_bacteria,Euryarchaeota,Desulfobacterota,Rikenellaceae_RC9_gut_group, andAlistipes than the CON phase (P < 0.05). The relative abundance ofBlautia in the HGE phase was higher than that in the HG phase (P < 0.05). The functional prediction results showed that SARA caused metabolic disorders in the dairy cows, while VE regulated the intestinal microbiota and health by improving the stability of microbial growth and promoting microbial reproduction.[Conclusion] The SARA induced by a high-concentrate diet led to reduced intestinal microbial diversity and metabolic disorders in dairy cows. VE can regulate intestinal health and maintain intestinal homeostasis by promoting the proliferation of beneficial intestinal microorganisms.

[Objective] This study aims to improve the bactericidal ability of phages against carbapenem-resistantKlebsiella pneumoniae, a major pathogen in clinical practice, and reduce host resistance to phages by developing a novel method of phage training.[Methods] Phages were isolated from municipal wastewater by the double-layer agar plate method with clinical carbapenem-resistantK.pneumoniae strain Kp2092 as the host, and their host ranges were analyzed. The morphological and genetic characteristics of a phage strain with strong lysis ability and a broad host range were analyzed by transmission electron microscopy and whole-genome sequencing. Phage training was performed by phage-host co-culture for generations, and the phages before and after training were compared in terms of biological properties such as bactericidal activity, optimal multiplicity of infection, one-step growth curve, and stability under different stress conditions.[Results] A total of 9 phage strains were isolated, in which P55anc was a short-tailed phage with the strongest lysis ability and the broadest host range. The genome (40 301 bp) of P55anc included 51 coding sequences, of which 27 showed possible functions involving nucleic acid metabolism, virion morphogenesis, DNA packaging, and host lysis. Three evolved phages of P55anc were obtained after 9 days of phage training. These evolved phages showed significantly enhanced bactericidal activities, manifested by the increased burst size, broadened host range, and reduced host resistance. Moreover, the evolved phages maintained stable when being exposed to heat, ultraviolet, and serum treatments.[Conclusion] Phage training by phage-host co-culture can be employed to obtain evolved phages with enhanced bactericidal effects. Furthermore, the evolved phages reduced the host resistance and remained stable under different stress conditions.

[Objective] Streptomyces is a genus of Gram-positive aerobic bacteria characterized by complex morphological differentiation and potent secondary metabolite-producing ability. SapB, a class Ⅲ lanthipeptide, promotes the morphological differentiation ofStreptomyces coelicolor, which suggests that SapB-like peptides might be developed as targets for engineering of morphological differentiation. In this study, we characterized the effects of SapB-like peptides on the morphological differentiation of multipleStreptomyces species, aiming to provide a theoretical basis for the engineering of these peptides.[Methods] Bioinformatics tools were used to analyze the gene clusters for the synthesis of SapB-like peptides in the genomes ofStreptomyces spp.. The plasmids for heterologous expression were constructed and introduced intoStreptomyces spp. through conjugation. The colony and mycelial morphology were compared to reveal the effects of these peptides on the morphological differentiation ofStreptomyces.[Results] SapB-like peptides promoted the differentiation ofStreptomyces from vegetative to aerial mycelia. Specifically, they increased the aerial mycelia and accelerated the differentiation, thus shortening the morphological differentiation cycle.[Conclusion] SapB-like peptides can help shorten the morphological differentiation cycle ofStreptomyces, demonstrating the potential for the morphological differentiation engineering ofStreptomyces.