[Objective] To determine the functions of two key enzymes of the mevalonate pathway, phosphomevalonate kinase (CmErg8) and mevalonate diphosphate decarboxylase (CmErg19), inCordyceps militaris and their effects on the content of ergosterol and cordycepin.[Methods] Bioinformatics analysis was conducted to identifyCmErg8 andCmErg19 inC.militaris, and yeast complementation was employed to determine whether their functions were conserved. Furthermore, we employedAgrobacterium tumefaciens-mediated transformation to overexpressCmErg8 andCmErg19 in the auxotrophic mutantCmΔpyrG ofC.militaris, so as to observe the effects ofCmErg8 andCmErg19 on the content of ergosterol and cordycepin.[Results] CmErg8 andCmErg19 could not complement the temperature sensitivity of theerg8 anderg19 mutants of yeast. The strains overexpressingCmErg8 andCmErg19 showed increased content of ergosterol and cordycepin. Particularly, the cordycepin content increased by about 5 times in the strain overexpressingCmErg19 compared with that in the control.[Conclusion] This study revealed the functions ofCmErg8 andCmErg19 inC.militaris and reported that the genes in the ergosterol synthesis pathway ofC.militaris affected the content of cordycepin for the first time.

[Objective] Pseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death. The emerging multi-drug resistant and pan-drug resistant strains pose challenges to the prevention and control ofP.aeruginosa infection.[Methods] A phage capable of lysing multi-drug resistantP.aeruginosa was isolated from farm sewage. The morphological characteristics, biological characteristics, host range, genomic features, andin vitro inhibitory ability of the isolate were studied. The biofilm inhibition test was conducted with both the phage and antibiotics.[Results] The phage PH826 was identified to be a member of the genus Nankokuvirus and it can keep stability within a wide temperature range from 4 ℃ to 60 ℃ and pH range (pH 3.0–11.0). The host range test showed that PH826 could lyse 13P.aeruginosa strains from human and animals. The antibacterial testin vitro showed that PH826 was strongly lytic againstP.aeruginosa at multiplicity of infection (MOI) of 10, 1, and 0.1. The genome of PH826 was 87 956 bp in length, with the G+C content of 54.70%, encoding 165 open reading frames (ORFs) including an endolysin gene. The combination of PH826 at 1×MIC (minimum inhibitory concentration, MIC), 2×MIC, or 4×MIC and ciprofloxacin reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. The combination of PH826 at 2×MIC or 4×MIC and meropenem reduced biofilm formation by more than 80% at the time points of 24 h and 48 h.[Conclusion] PH826 may be a potential disinfectant and therapeutic agent that can be used alone or in combination with antibiotics for the prevention and treatment of pathogenicP.aeruginosa.

[Objective] To construct the γ-aminobutyrate-producing recombinant strains ofEscherichia coli and investigate their fermentation characteristics.[Methods] We constructed two recombinant plasmids pTrc99a-gadB and pTrc99a-gadB-SNO1-SNZ1 and then respectively transformed them into the gene-knockout strainE.coli K12/ΔgabTΔgabPΔpuuE. We investigated and optimized the fermentation process of the recombinant strains for producing γ-aminobutyrate.[Results] The target proteins were highly expressed in the recombinant strains harboring the constructed plasmids. The highest concentration of γ-aminobutyrate was 4.6 g/L in the fermentation broth ofE.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB cultured in the medium containing 10 g/L l-monosodium glutamate and was 21.9 times higher than that in the fermentation broth of the wild type strain. At the l-monosodium glutamate concentration of 20 g/L, the conversion rate of substrate was the highest and the concentration of γ-aminobutyrate reached 8.4 g/L. The concentration of γ-aminobutyrate was slightly lower in the fermentation broth of the recombinant strainE.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1, probably due to the excessive consumption of energy. The highest concentration of γ-aminobutyrate was 9.4 g/L whenE.coli K12ΔgabTΔgabPΔpuuE/ pTrc99a-gadB was cultured in 1 L fermentation medium containing 20 g/L l-monosodium glutamate.[Conclusion] We obviously increased the yield of γ-aminobutyrate produced by the recombinant strain. This finding lays a foundation for the industrial production of γ-aminobutyrate.

[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 increasing antibiotic resistant bacteria (ARB) caused by the horizontal transfer of antibiotic resistance genes (ARGs) have aroused increasing concern, and it is particularly important to monitor the ARB in the environment.[Methods] We collected seawater and sand samples from three different bathing beaches in Zhanjiang. The plate coating method and agar diffusion method were employed to study the bacterial counts, diversity, and susceptibility to antibiotics.[Results] The seawater samples had higher content of inorganic nitrogen than the normal value and the bacterial counts increased with the increase in the visitor flow. The bacteria counts in the sand samples were higher than those in the seawater samples. The bacteria from the bathing beaches belonged into 18 genera, 12 families of 3 phyla.Proteobacteria (49.64%) was dominant in the seawater samples, whileFirmicutes (54.74%) was dominant in the sand samples. The bacteria showed stronger resistance to beta-lactams, with the resistance rates of 23.25%, 20.53%, and 17.42% to penicillin, vancomycin, and ceftriaxone, respectively. The resistant bacteria were mainlyBacillus,Vibrio,Pseudomonas,Streptomyces, andEnterobacter. The multidrug-resistant bacterial strains from seawater were significantly more than those from sand, and increased with the increase in the visitor flow.[Conclusion] There are diverse drug-resistant bacteria from bathing beaches, and continuous surveillance is required to assess its potential impact on public health.

[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] We isolated bacteria from the rhizosphere soils of three blueberry varieties, explored the bacterial diversity, and screened out the strains with acid-producing, plant growth-promoting, and antifungal properties, aiming to provide high-quality strain resources and a theoretical basis for the research on blueberry-specialized microbial fertilizers.[Methods] Five different media were used to isolate the bacteria from rhizosphere soils, and 16S rRNA gene sequencing and phylogenetic analysis were performed. The strains capable of producing acid, indole-3-acetic acid (IAA), and siderophores, fixing nitrogen, solubilizing phosphorus, and inhibiting the growth ofBotrytis cinerea were screened out. The suitable strains with excellent properties were selected and then inoculated in blueberry seedlings cultivated in pots. The effects on the growth and element absorption of the seedlings and fertility of rhizosphere soil were examined.[Results] A total of 124 strains were isolated from the rhizosphere soils of three blueberry varieties. Seventy representative strains were selected for 16S rRNA gene sequencing, belonging to 21 genera of 3 phyla, among whichBacillus,Pseudomonas,Streptomyces, andRhodococcus were the dominant bacteria. Among the representative strains, 21.4%, 21.4%, 47.1%, 65.7%, and 14.3% could produce acids, produce IAA, fix nitrogen, solubilize phosphorus, and secrete siderophores, respectively. A few strains displayed the abilities of producing acids and IAA, fixing nitrogen, solubilizing phosphorus, and inhibitingB.cinerea simultaneously.Pseudomonas chlororaphis CSM-70 andPseudomonas piscium CSM-129 with acid-producing and growth-promoting characteristics were selected and inoculated in blueberry seedlings. Both strains significantly promoted the growth and development of blueberry seedlings and regulated the pH of the rhizosphere soil. In addition, strain CSM-70 significantly promoted the absorption of nitrogen and phosphorus in blueberry leaves and increased the content of available potassium and available nitrogen in the soil.[Conclusion] The blueberry rhizosphere soil has high bacterial diversity and harbors abundant plant growth-promoting strains.P.chlororaphis CSM-70 andP.piscium CSM-129 can promote the growth of blueberry seedlings, regulate rhizosphere soil pH and fertility, and promote nutrient absorption, demonstrating the potential of serving as blueberry-specific microbial fertilizers.

[Objective] The long-term intense continuous cropping and abuse of mineral fertilizers result in the degradation of upland red soil and the accumulation of soil-borne plant pathogens. Fungi are the microorganisms closely related to soil health in agroecosystems. We investigated the changes of soil fungal community to explore the effects of hairy vetch (Vicia villosa Roth L.) application on the agroecosystem with upland red soil.[Methods] We employed quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (Illumina MiSeq) to investigate the responses of fungal communities to mineral fertilizer (hereinafter referred to as NPK) alone and mineral fertilizer combined with hairy vetch (hereinafter referred to as NPKG) in upland red soil.[Results] Compared with NPK, NPKG increased the soil fertility, peanut yield, and fungal abundance and decreased soil pH and soil fungal diversity. Different treatments significantly altered the soil fungal community composition. Compared with NPK, NPKG increased the relative abundance of saprophytic fungi by 37.42% and decreased the relative abundance ofCercospora arachidicola andLasidiplodia theobromae by 89.11% and 88.10%, respectively.[Conclusion] The application of hairy vetch significantly increased soil fertility, reduced the risk of peanut exposure to soil-borne diseases, and increased peanut yield in the upland red soil. Therefore, the application of hairy vetch was conducive to the sustainable development of upland red soil in southern China.

[Objective] To study the transcriptional regulation of type Ⅵ secretion system 1 (T6SS1) genes by QsvR inVibrio parahaemolyticus.[Methods] Total RNA was extracted from the wild type (WT) andqsvR mutant (ΔqsvR). Quantitative real-time PCR (qPCR) was employed to investigate the transcriptional regulation of target genes by QsvR. Primer extension was carried out to detect the transcription initiation site and core promoter for each target gene and calculate the transcriptional variations between WT and ΔqsvR. The regulatory DNA region of each target gene was cloned into the restriction endonuclease sites of pHRP309 harboring a promoterless genelacZ, and then each recombinant plasmid was transferred into WT and ΔqsvR, respectively. A β-Galactosidase Enzyme Assay System (Promega) was used to measure the β-galactosidase activity in cell lysates. The recombinant pHRP309 vector containing the regulatory DNA region of one of the target gene was transferred intoEscherichia coli 100λpir harboring an empty pBAD33 or pBAD33-qsvR to test whether QsvR can regulate the target genes in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and His-QsvR was over-expressed and then purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-binding activity of His-QsvR to each target DNA fragmentin vitro.[Results] The mRNA levels of T6SS1-associated genes, VP1388 (the first gene of VP1388–1390 operon) andhcp1 (the first gene of VP1393–1406 operon), were significantly up-regulated in ΔqsvR relative to those in WT, indicating that QsvR activated the transcription of VP1388 andhcp1. Only one transcription initiation site was detected for VP1388 orhcp1, locating at 64 bp upstream of VP1388 and 62 bp upstream ofhcp1, respectively, and their transcriptional activities were all repressed by QsvR. QsvR repressed the promoter activities of VP1388 andhcp1 in bothV.parahaemolyticus andE.coli 100λpir. His-QsvR was able to bind to the regulatory DNA regions of VP1388 andhcp1.[Conclusion] QsvR directly repressed the transcription of T6SS1-associated operons, VP1388–1390 and VP1393–1406, inV.parahaemolyticus.