[Objective] To study the effects of different water and fertilizer conditions on the biomass of safflower and the phosphorus and microorganisms in the rhizosphere soil, and isolate the strains with high phosphorus-solubilizing ability from the rhizosphere soil samples, so as to provide a theoretical basis for the reasonable water and fertilizer management of safflower and excellent strains for the research on safflower growth and rhizosphere microenvironment.[Methods] The safflower plants were treated with phosphorus fertilizer at different gradients, and the plant biomass was determined at the rosette, jointing, blooming, and seed maturity stages. At the same time, the microbial community composition, total phosphorus, available phosphorus, and phosphatase activity in the rhizosphere soil were determined. The strains with high phosphorus-solubilizing ability were screened by soil shaking and dilution coating methods and preliminarily identified by comparative analysis of 16S rRNA gene sequences. The phosphorus-solubilizing abilities of the strains in different media were determined by the molybdenum antimony colorimetric method. Furthermore, we inoculated the dominant strains by using the root irrigation method and dilution coating method to analyze the abilities of the strains to colonize the rhizosphere and promote the growth of safflower.[Results] W3-P2 water and fertilizer treatments were conducive to the biomass accumulation of safflower. As the application amount of phosphorus fertilizer increased, the available phosphorus content and phosphatase activity showed a trend of first increasing and then decreasing. The effects of water on soil total phosphorus, available phosphorus, and phosphatase activity were correlated with the safflower development stages. Bacteria were the dominant microorganisms in the rhizosphere soil of safflower, with the highest counts of 3.017×10⁷ CFU/g and 3.021×10⁷ CFU/g at the seed maturity stage in the water treatment and phosphorus fertilizer treatment groups, respectively, which were much higher than those of fungi and actinomycetes in the same treatment group. Five efficient phosphorus-solubilizing strains (accession number C1: OR493125; C2: OR493126; C5: OR493127; C6: OR493128; C7: OR493129) were screened out from the rhizosphere soil of safflower. All of them had the ability to solubilize phosphorus and lowered the pH of the medium with inorganic and organic phosphorus as the only phosphorus source. Strain C6 had the strongest ability to solubilize phosphorus, with the amounts of phosphorus solubilized in the inorganic phosphorus media of tricalcium phosphate, aluminum phosphate, ferric phosphate, and calcium phytate being 380.00, 269.32, 7.15, and 48.16 mg/L, respectively. It solubilized 18.19 mg/L phosphorus in the medium with organic phosphorus (lecithin) as the only phosphorus source. C6 was identified by 16S rRNA gene sequencing analysis asPseudomonas sp. and C1, C2, C5, and C7 asSinorhizobium sp. After inoculation of 2% suspensions (10⁸ CFU/mL) of the dominant phosphorus-solubilizing bacterial strains C1, C5, and C6 around safflower plants, the cell count remained 10⁵ CFU/g on day 21, and C6 demonstrated stronger colonization. The strains increased the leaf number, plant height, stem diameter, stem weight, and root length of safflower at the blooming stage, and the C6 strain group presented stronger effects, with the indicators above being 122 tablets, 115.96 cm, 12.49 mm, 43.36 g, and 21.17 cm, respectively.[Conclusion] Water and fertilizer management affects the available phosphorus content and microbial community structure in the rhizosphere microenvironment of safflower and promotes the growth and development of safflower roots, thus directly or indirectly affecting the biomass of safflower. The irrigation at 3 000 m³/hm² and phosphorus fertilizer application 375 kg/hm² were suitable for the growth of safflower. C6 is an efficient phosphorus-solubilizing strain, capable of decomposing insoluble organic and inorganic phosphorus. Pot experiments showed that C6 could colonize the rhizosphere of safflower and significantly promoted the growth of safflower.

[Objective] Cytochrome P450 can catalyze the oxidation of unactivated C−H bonds in complex compounds with high regio- and stereoselectivity under mild conditions. They are multifunctional biocatalysts which play important roles in the synthesis of chemical raw materials, the degradation of environmental pollutants, and drug synthesis. This paper probed into the functions of a novel cytochrome P450 enzyme named CYP154C34 fromStreptomyces nanshensis by heterologous expression and whole-cell biotransformation.[Methods] We constructed two recombinant BL21(DE3) strains for whole-cell biotransformation harboring pET28a-CYP154C34-RhFRED and pET28a-CYP154C34+pACYCDuet-Pdx/PdR, respectively. Additionally, we generated a recombinant BL21(DE3) strain for heterologous expression containing pET28a-CYP154C34. We employed whole-cell biotransformation to screen the substrates and analyzed product structures using standard methods. We then compared the substrate conversion rates of the two strains of whole-cell biotransformation with those of enzyme reactions. Finally, we analyzed the substrate affinity and analogues of CYP154C34.[Results] Nine steroids hydroxylated at the 16α position by CYP154C34 were identified, including progesterone, testosterone, and androstenedione. The BL21(DE3) strain carrying pET28a-CYP154C34-RhFRED showed the highest substrate conversion rate, achieving over 90% conversion rates for seven substrates including progesterone, testosterone, and androstenedione. In addition, CYP154C34 had stronger affinity to its substrates than the analogues.[Conclusion] This study constructed two recombinant strains of CYP154C34 for whole-cell biotransformation and identified their catalytic functions. CYP154C34 can efficiently hydroxylate a wide range of steroid substrates at the 16α position with high conversion rates and excellent regio- and stereoselectivity, serving as a promising candidate for industrial applications.

[Objective] To investigate the effect of supplementing a probiotic mixture (Bacillus licheniformis,B.subtilis, andClostridium butyricum) in drinking water on the meat quality of broilers and explore the underlying mechanism.[Methods] A total of 360 1-day-old white feather broilers were randomly selected and assigned to control (CON, normal drinking water), low-dose probiotic (LG, drinking water supplemented with 0.2% probiotic mixture), and high-dose probiotic (HG, drinking water supplemented with 0.4% probiotic mixture) groups. The experiment lasted for 42 days.[Results] Compared with the CON group, the LG and HG groups showed increased average body weight on days 7, 35, and 42. The HG groups increased average daily gain during days 21–28, 28–35, and 35–42, while the LG groups increased average daily gain during days 35–42. LG and HG significantly increased the chest muscle redness at the time points of 45 min, 24 h, and 48 h and the chest muscle yellowness at the time point of 24 h, decreased the brightness at 24 h and 48 h and the dripping and cooking loss at 48 h. The HG group had higher content of crude protein and crude fat in chest muscle than the CON group and no significant difference compared with the LG group. Both LG and HG groups reduced the ash content of chest muscle. The addition of the probiotic mixture significantly increased total antioxidant capacity (T-AOC), while the levels of total-superoxide dismutase (T-SOD) and catalase (CAT) had an increasing trend. The expression levels ofCAT,SOD-1,Keap1, andNrf2 were significantly up-regulated, and the HG group had higher expression levels ofSOD-2 andHO-1 than the LG group. The concentrations of acetic acid, propionic acid, butyric acid, and valeric acid in cecum on days 21 and 42 significantly increased in LG and HG groups.[Conclusion] The probiotic mixture enhanced the growth performance and meat quality of broilers by increasing the levels of antioxidant activity and short-chain fatty acids, and the HG group outperformed the LG group.

[Objective] The cell wall structure characteristics make it a challenge to obtain the whole proteins ofPaenibacillus sp. as a Gram-positive bacterium. We employed three representative lysis methods: lysozyme combined with ultrasonic treatment (method 1), lysozyme combined with SDS heat treatment (method 2), and liquid nitrogen combined with ultrasonic treatment (method 3), to obtain the samples for mass spectrometry-based proteomics research onPaenibacillus sp.[Methods] We compared the protein extraction efficiency of the three methods based on protein yield and SDS-PAGE. Furthermore, we analyzed the obtained protein samples by mass spectrometry.[Results] The protein extraction efficiency of the three methods was similar. The number of proteins identified in the sample prepared with method 3 was the highest (2 638 proteins), followed by that with method 1 (2 452 proteins), and that with method 2 was the lowest (2 003 proteins). The Venn diagram was established to compare the protein identification flux of the samples prepared with different methods. Considering the protein extraction efficiency and the shortcomings of method 3, we finally chose method 1 as the most suitable method to extract the proteins for mass spectrometry-based proteomic analysis. Finally, the basic properties of proteins identified by mass spectrometry, including molecular weight, isoelectric point, and hydrophobicity, were analyzed. The proportion of each component of the proteins obtained by the three methods was the same as that of the predicted proteins of the model strainPaenibacillus polymyxa.[Conclusion] For the proteomics research on Gram-positivePaenibacillus sp. by mass spectrometry, lysozyme combined with ultrasonic treatment is the most suitable method for sample preparation.

Guanosine tetraphosphate (ppGpp)/guanosine pentaphosphate (pppGpp) is a signaling molecule in the bacterial stringent response, whose synthesis and hydrolysis are controlled by the synthesis and hydrolysis activities of proteins in the RelA/SpoT homologue (RSH) family. The (p)ppGpp-mediated stringent response enhanced bacterial adaptation to nutrient deprivation and antibiotic resistance. In recent years, (p)ppGpp has been found to associate with bacterial growth, cell division, and antibiotic synthesis, which was an important global regulator in the bacteria. (p)ppGpp has many target sites in the cell, which allow it to regulate DNA replication, transcription, cell cycle, ribosome biosynthesis, and the expression of antibiotic synthesis gene clusters. However, how (p)ppGpp controls transcription and other metabolic processes depends on the bacterial species, and (p)ppGpp regulates the same processes in different bacteria species through different mechanisms. Therefore, this manuscript reviewed the types of (p)ppGpp synthetic and hydrolytic enzymes, the mechanisms of (p)ppGpp regulation on microbial metabolism and the cell cycle, as well as the regulation mechanisms of antibiotic synthesis and tolerance, which lays the foundation for bacterial resistance and cell physiology researches.

With the evolution of bacteria and the overuse of certain antibiotics, drug-resistant bacterial infections have emerged as a major public health challenge in the 21st century.Klebsiella pneumoniae, in particular, has aroused wide concern due to its drug-resistant nature. Bacteriophages have demonstrated the potential and advantages in treating bacterial infections caused by drug-resistant strains, while there are no clinical guidelines for phage therapy. Notably, even though a few successful cases of bacteriophage therapy have been documented in the treatment ofK.pneumoniae infections, bacteriophages are used in combination with antibiotics in most cases and the role bacteriophages play remains unclear. This article provides an overview of bacteriophage therapies forK.pneumoniae infections, including their characteristics and the factors influencing their efficacy. We compiled and analyzed data from available studies, with the intention of offering valuable insights for the application of bacteriophage therapy in combatingK.pneumoniae and other drug-resistant bacteria.

[Objective] To investigateBacillus velezensis XRD006 in terms of the inhibitory effects on diseases of postharvest green walnuts, the fresh-keeping effect on green walnuts, genetic characteristics, secondary metabolites, and antifungal mechanism.[Methods] The activities of XRD006 against pathogens of postharvest green walnuts were determined by the inhibition experiments.In vivo fungal inhibition and storage quality experiments were conducted to investigate the inhibitory effects of the strain on pathogens and the effect of the strain on the storage quality of postharvest green walnuts. The genomic characteristics and potential antifungal genes of XRD006 were investigated by whole genome sequencing. The secondary metabolites of XRD006 were predicted by antiSMASH, and the collinearity and differences of the secondary metabolite gene clusters between strains XRD006, FZB42, and SQR9 were analyzed by comparative genomics. The secondary metabolites of XRD006 were identified by high performance liquid chromatography (HPLC) and mass spectrometry. The antagonistic ability of the strain was tested by the oxford cup method.[Results] The inhibition rates of XRD006 againstColletotrichum aenigma,Colletotrichum siamense,Botryosphaeria dothidea, andFusarium fujikuroi of postharvest green walnuts were 49.22%, 50.61%, 53.83%, and 58.71%, respectively.In vivo antifungal experiments showed that XRD006 effectively inhibited the infection and growth of pathogenic fungi on the fruits. The fermentation supernatant of XRD006 significantly retarded the weight loss, inhibited the microbial growth, and reduced changes in peroxidase (POD) and polyphenol oxidase (PPO) activities while maintaining the kernel quality. The whole genome sequencing showed that the genome of XRD006 was 4 371 975 bp in length, containing 46.07% GC and 4 362 protein-coding genes (including antifungal and plant growth-promoting genes such as extracellular hydrolase and biofilm genes). The antiSMASH predicted that XRD006 had nine known and five unknown gene clusters of secondary metabolites. XRD006 was closely related to FZB42 and SQR9, and they shared eight secondary metabolite gene clusters, which showed varied location and coding genes. XRD006 produced two families of lipopeptides: iturin and fengycin. Compared with C13-iturin, C14-iturin, C15-iturin, and C17-fengycin C, C16-fengycin B of the fengycin family had the strongest inhibitory effect onC.siamense HT12.[Conclusion] B.velezensis XRD006 has good biocontrol effects on diseases of postharvest green walnuts and the potential for application in production.

[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] 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 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.