[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] 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 mitigate the threat of heavy metal pollution in wastewater to global food safety and human health, reduce the accumulation of lead (Pb) in soil, plants, and animals, and improve the removal rate of heavy metals by immobilizing microbial strains.[Methods] We carried out mixed strain test to select the white rot fungal strains with strong Pb²⁺ removal effects and excellent compatibility and explored the optimal strains and ratio for combination. Furthermore, we optimized the formula of the fungal strain composite and explored the optimal adsorption conditions of the composite in application.[Results] Phanerochaete chrysosporium,Coriolus versicolor,Lentinus sajor-caju, andPleurotus ostreatus with good compatibility were selected for subsequent experiments.C.versicolor mixed withL.sajor-caju at a volume ratio of 1:1 outperformed the single strains in removing Pb²⁺. The fungal strain composite composed of 20.0 g/L sodium alginate, 15.0 g/L biochar, 2.0×10⁶ CFU/mL white rot fungi, silica, and zeolite showed the Pb²⁺ removal rate of 90.63% within 96 h. Moreover, this composite had higher mechanical strength and strong resistance to mechanical shear. At the addition amount of 8.35 g/L and pH 5.64, the composite demonstrated the Pb²⁺ removal rate of 97.45% within 96 h. Moreover, this composite can be reused 7 times after adsorption-desorption-readsorption and maintained high Pb²⁺ removal capacity.[Conclusion] The immobilized white rot fungal strain composite can significantly improve the microbial utilization rate and wastewater treatment efficiency compared with single strains. It can greatly adsorb Pb²⁺ in wastewater under appropriate conditions within a short time and reduce the environmental threat caused by heavy metal pollutants. Therefore, the promotion of environmental protection greatly benefits from the use of immobilized mixed white rot fungal strains in the treatment of heavy metal-contaminated wastewater.

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 thuringiensis (Bt), characterized by the massive production of insecticidal crystal proteins (ICPs) during sporulation, serves as the main strain resource for the commonly used and safe microbial insecticides. To further explore the mechanisms of sporulation and parasporal crystal formation and lay a theoretical foundation for the construction of efficient strains, we compared the transcriptomes of Bt at three important stages.[Methods] The transcriptomes of the hypervirulent strain Bt4.0718 at the middle vegetative growth stage (T1-10 h), the early sporulation stage (T2-20 h), and the late sporulation stage (T3-32 h) were compared. The representative differentially expressed genes (DEGs) were verified by real-time fluorescence quantitative PCR (qRT-PCR), and the phenotypes of the mutant strains with the knockout of specific functional genes were examined.[Results] The number of DEGs was 2 147 (T2/T1), 1 861 (T3/T1), and 1 708 (T3/T2), respectively. At T1, the medium was rich in nutrients, which served the sporulation and parasporal crystal formation. The high transcription levels ofkinA/D,spo0A/F, andsigE regulating sporulation played a role in the growth and development of the cells. The transcription of Cry1Ac, poly-hydroxybutyric acid (PHB), and hydroxybutanone (acetoin) were started at this time. The substantial formation of ICPs and spores occurred at T2 and T3, and the transcript levels of the regulatory genes were higher at T2 than those at T3. The genes associated with spore core/coat/cortex, germination protein, andspoII–spoVI began to be transcribed in large amounts at T2, with the highest levels among the three stages. The corresponding complex networks of carbohydrate, amino acid, and lipid metabolism, energy, nucleic acid, and peptide metabolism, secondary metabolite production, and environmental adaptation showed differences. In addition, as the physiological processes stimulated by nutrient signals, the two-component signal transduction system (TCS) and ABC transport system played an essential role in the process of sporulation and ICP transcription and expression, and their transcription levels were significantly different.[Conclusion] With the production of ICPs and sporulation, nutrients are gradually consumed, and the high expression ofsigB,sigW, andsigM contributed to the stability of cell wall and the resistance to environmental changes. Meanwhile, the small heat shock proteins Hsp20 and Hsp20B, as molecular chaperones, were also important for maintaining intracellular homeostasis and may facilitate the sporulation and ICP production.

[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.

Fatty acids are not only the components of cell membrane but also the raw materials for the synthesis of bioactive molecules. Unsaturated fatty acids (UFAs) with low phase-transition temperatures are essential molecules for bacteria to regulate cell membrane fluidity. Therefore, the synthetic pathways of UFAs are key targets for the screening of antibacterial agents. Bacteria can adopt the anaerobic pathway to synthesize UFAs. For example,Escherichia coli, a model organism, synthesizes UFAsvia the classic FabA-FabB pathway. However, the anaerobic pathways for the synthesis of UFAs vary in different bacteria, and the catalytic enzymes are also different. Bacteria can synthesize UFAsvia aerobic pathways, in which fatty acid desaturase directly converts saturated fatty acids (SFAs) into UFAs. Different desaturases introduce double bond to form UFAs with different structures, which play roles in stress responses, pathogenicity and other aspects. Other enzymes involved in the synthesis of fatty acids can also participate in the synthesis of UFAs or regulation of different UFAs. Some bacteria can use monooxygenase to convert capryl-ACP (acyl carrier protein) in the fatty acid synthesis pathway intocis-3-decenyl ACP to synthesize UFAs. We comprehensively reviewed the research progress in the synthesis of UFAs in bacteria, aiming to provide theoretical support for deciphering the mechanism of bacterial synthesis of UFAs and developing the targeted antibacterial drugs.

Proper pH is crucial for the survival and functions of microorganisms, whether in the environment or within cells. Under acidic or alkaline stress, microorganisms have evolved diverse strategies, such as proton transport, production of acidic or alkaline substances, and cell membrane protection, to maintain intracellular pH homeostasis. Moreover, microorganisms have evolved the ability to actively change the extracellular pH. This article reviews the mechanisms by which microorganisms maintain intracellular pH homeostasis under acid or alkaline stress and alter extracellular pH. It aims to enhance our understanding of the interaction between microorganisms and the environment and provide a reference for further research on the synergistic mechanisms between microorganisms and the environment.