Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Isolation and characterization of Stenotrophomonas pavanii GXUN74707 with efficient flocculation performance and application in wastewater treatment.

The identification of microorganisms with excellent flocculants-producing capability and optimization of the fermentation process are necessary for the wide-scale application of bioflocculants. Therefore, we isolated and identified a highly efficient flocculation performance strain of Stenotrophomonas pavanii GXUN74707 from the sludge. The optimal fermentation and flocculation conditions of strain S. pavanii GXUN74707 was in fermentation medium with glucose and urea as the carbon and nitrogen sources, respectively, at pH 7.0 for 36 h, which treatment of kaolin suspension with 0.5 mL of the fermentation broth resulted in a flocculation rate of 99.0%. The bioflocculant synthesized by strain S. pavanii GXUN74707 was found mainly in the supernatant of the fermentation broth. Chemical analysis revealed that the pure bioflocculant consisted of 79.70% carbohydrates and 14.38% proteins. The monosaccharide components of MBF-GXUN74707 are mainly mannose (5.96 µg/mg), galactose (1.86 µg/mg), and glucose (1.73 µg/mg). Infrared spectrometric analysis showed the presence of carboxyl (COO-), hydroxyl (-OH) groups. The SEM images showed clumps of rod-shaped bacteria with adhesion of extracellular products. Furthermore, the strain decolored dye wastewater containing direct black, direct blue, and Congo red by 89.2%, 95.1%, 94.1%, respectively. The chemical oxygen demand (COD) and biological oxygen demand (BOD) removal rates after treatment of aquaculture wastewater with the fermentation broth were 68% and 23%, respectively. This study is the first to report the performance and application of strain Stenotrophomonas pavanii in wastewater flocculation. The results indicate that strain S. pavanii is a good candidate for the production novel bioflocculants and demonstrates its potential industrial practicality in biotechnology processes.

First Report of Diaporthe hongkongensis Causing Leaf Blight on Macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) belongs to the Proteaceae family (Li et al. 2022). In the hilly areas of Guangxi (southern China), macadamia trees are an important source of revenue. The planting area in Guangxi has increased in recent years, exceeding 53,333 hectares by the end of 2022, but this increase is also associated with emergency of, macadamia diseases. Leaf blight symptoms were observed in 37/241 macadamia trees (15% incidence) in a plantation in Nanning, Guangxi province in China, during June, 2022. Disease severity on infected trees ranged from 5% to 60%. The disease developed from the tips or margins of leaves, causing the leaves to turn brown, and later gradually withered (Fig. 1 A). Ten leaves with lesions were collected from five macadamia trees (two leaves per tree. Thereafter, small segments (3 to 4 mm²) excised from the margins of ten lesions were surface sterilized in 75% ethanol for 30 s and 1% hypochlorite for 90 s and Page 1 of 6 2 rinsed in sterile water, before plating onto potato dextrose agar (PDA) medium. Plates were incubated under lighting during the daytime, and darkness at night-time for 5 days at 25℃. Twenty-two purified colonies were generated by subculturing hyphal tips, of which eight exhibited similar morphology and were further characterized. The colonies on PDA were gray with a white outer ring and flat lawn on the surface (Fig. 1 B). The pycnidia were superficial to semi-immersed on PDA, solitary to aggregated, globose to sub-globose, brown to black and oozed yellow mucilaginous masses (Fig.1 C). The α-conidia were unicellular, hyaline elliptical or fusiform, and measuring 4-8 × 1.9-4 µm (n=30) ， whereas the ß-conidia were hyaline, long, straight or curved, measuring 20-23 × 0.9-2 µm (n=30) (Fig. 1 D-E). The morphological features were similar to Diaporthe hongkongensis (Dissanayake et al. 2015). The eight morphologically similar isolates were identified as D. hongkongensis using the internal transcribed spacer (ITS) region, but only one isolate, JG11, was selected for further molecular identification. Five target genes, including the ITS region, translation elongation factor 1 alpha (EF1-α), beta-tubulin genes (TUB2), calmodulin (CAL), and histone H3 (HIS) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, CAL-228F/CAL-737R, and CYLH3F/H3-1b, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank under accession numbers OQ932790 (ITS) and OR147955-58 for EF1-α, TUB, CAL and HIS genes, respectively. BLAST search of GenBank showed that ITS, EF1-α, TUB, CAL, and HIS sequences of JG11 were similar to Page 2 of 6 3 those of D. hongkongensis NR111848 (99.22% identity), KY433566 (99.72%), MW208603 (99.42%), MW221740 (99.80%), and MW221661 (99.79%), respectively. Phylogenetic analysis of concatenated sequences was performed with IQ-TREE software. JG11 was grouped in the same clade as other Diaporthe hongkongensis isolates (Fig. 2). Pathogenicity experiments were carried out on healthy macadamia trees in a greenhouse. Three macadamia trees were used as negative controls where five uninjured leaves per tree were sprayed with sterile distilled water. Uninjured five leaves per tree of three other macadamia trees were sprayed with conidia suspension of the isolate JG11 at a concentration of 1×106. Each treatment was repeated 3 times independently, with 5 leaves per tree (Liu et al. 2023; Havill et al. 2023; Zhang et al. 2022). Plastic bags were placed over all inoculated leaves. The average daily temperature and relative humidity in the greenhouse were 32°C and 65%, respectively. Two days later, browning appeared on the leaves inoculated with the spore suspension and expanded outward. After 5 days, all macadamia leaves inoculated with the fungal spores began to wither, while controls remained asymptomatic (Fig. 1 H-I). D. hongkongensis was consistently re-isolated and purified from inoculated leaves and the identity was confirmed by morphological identification and molecular analysis, completed Koch's postulates. D. hongkongensis has been reported on peach (Zhang et al. 2021), grapevine trunk (Dissanayake et al. 2015) and Cunninghamia lanceolata (Liao et al. 2022). To our knowledge, this is the first report of D. hongkongensis causing leaf blight on macadamia in China. These findings provide a foundation for future research on the epidemiology and control of this newly emerging disease of macadamia.

A Comparative Transcriptome Analysis Unveils the Mechanisms of Response in Feather Degradation by Pseudomonas aeruginosa Gxun-7.

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT-PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming.

Discovery of Antifungal Norsesquiterpenoids from a Soil-Derived Streptomyces microflavus: Targeting Biofilm Formation and Synergistic Combination with Amphotericin B against Yeast-like Fungi.

Thirty-five norsesquiterpenoids were isolated from the fermentation broth of Streptomyces microflavus from the forest soil of Ailaoshan in China. The structures of new compounds (1-5, 10-26) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra, as well as Mosher's reagent derivatives method. Norsesquiterpenoids showed different levels of antifungal activity with MIC80 values ranging from 25 to 200 µg/mL against Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. The combining isolated norsesquiterpenoids with amphotericin B resulted in a synergistic interaction against test yeast-like fungi with a fractional inhibitory concentration index < 0.5. Compound 33 significantly inhibited biofilm formation and destroyed the preformed biofilm of fungi. Moreover, 33 downregulated the expression of adhesion-related genes HWP1, ALS1, ALS3, ECE1, EAP1, and BCR1 to inhibit the adhesion of C. albicans. Findings from the current study highlight the potential usage of norsesquiterpenoids from soil-derived Streptomyces for antifungal leads discovery.

Nesterenkonia marinintestina sp. nov., isolated from the fish intestine.

A Gram-positive, aerobic, non-motile, irregular short rod, nonspore-forming actinobacterial strain, designated GX14115T, was isolated from fish intestine in Beihai City, Guangxi, China and subjected to a taxonomic polyphasic investigation. Colonies were yellow‒green, circular, smooth, central bulge, convex, opaque and 2.0-3.0 mm in diameter after growth on 2216E medium at 30 °C for 72 h. Growth occurred at 4-45 °C (optimum 30 °C), at pH 4.5-10.0 (optimum pH 7.5) and in the presence of 0-12% NaCl (w/v) (optimum 3.5%). Chemotaxonomic analysis showed that the main menaquinone of strain GX14115T was MK-7. The major cellular fatty acids were anteiso-C15:0 (44.8%), anteiso-C17:0 (20.5%), and iso-C15:0 (16%). The whole-cell sugars were galactose and xylose. The peptidoglycan type was L-Lys-Gly-D-Asp, and the polar lipids were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), one unknown phospholipid (UP), and one unknown glycolipid (UG). The DNA G + C content of the type of strain was 69.5 mol%. The 16S rRNA gene sequence analysis revealed that strain GX14115T is affiliated with the genus Nesterenkonia and is closely related to Nesterenkonia sandarakina YIM 70009T (96.5%) and Nesterenkonia lutea YIM 70081T (96.8%). The calculated results indicated that the average nucleotide identity (ANI) values of GX14115T were 74.49-74.78%, to the two aforementioned type strains, and the digital DNA-DNA hybridization (dDDH) values were 20.1-20.7%. Strain GX14115T was proposed as a novel species of the genus Nesterenkonia by the physiological, chemotaxonomic, and phylogenetic data, for whose the name is Nesterenkonia marinintestina sp. nov. The type of strain is GX14115T (= MCCC 1K06658T = KCTC 49495T).

Microbulbifer litoralis sp. nov., Isolated from Seashore of Weizhou Island.

A bacterium designated GXH0434T was isolated from sea shore samples collected from Weizhou Island, Beihai, Guangxi, China. The organism is motile, strictly aerobic, and possesses a rod-coccus cell cycle in association with the growth phase. It can grow at 15-45 °C (optimum 37 °C), at pH 6.0-11.0 (optimum 6.0), and at 0-20% (w/v) NaCl (optimum 5.0-8.0%). The strain is positive for peroxidase and oxidase activity, negative for Voges-Proskauer test, can hydrolyze Tween 20, Tween 60, Tween 80, casein, and is able to produce siderophore and has the function of nitrogen fixation. Molecular phylogenetic analysis based on 16S rRNA gene sequences indicated that GXH0434T was most closely related to Microbulbifer halophilus KCTC 12848T with the similarity of 97.2%, followed by Microbulbifer chitinilyticus JCM 16148T (97.1%) and Microbulbifer taiwanensis LMG 26125T (96.5%). The digital DNA-DNA hybridization and the average nucleotide identity values between GXH0434T and Microbulbifer halophilus KCTC 12848T were 28.90% and 83.38%, respectively, which were below thresholds of species delineation. The genomic DNA G+C content of the strain was 61.9%. The major fatty acids were iso-C15:0, C16:0, iso-C11:0 3-OH, iso-C11:0 and Summed features 8 (C18:0 ω7c and/or C18:0 ω6c). The major polar lipids detected in GXH0434T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC). The major respiratory quinone was ubiquinone Q-8. Based on the above polyphasic classification indicated strain GXH0434T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer litoralis sp. nov. is proposed. The type strain is GXH0434T (= MCCC 1K07158T = KCTC 92169T).

Marinimicrococcus flavescens gen. nov., sp. nov., a new member of the family Geminicoccaceae, isolated from a marine sediment of the South China Sea.

A Gram-stain-negative, catalase-positive and oxidase-positive, nonmotile, aerobic, light yellow, spherical-shaped bacterial strain with no flagella, designated strain YIM 152171T, was isolated from sediment of the South China Sea. Colonies were smooth and convex, light yellow and circular, and 1.0-1.5×1.0-1.5 µm in cell diameter after 7 days of incubation at 28°C on YIM38 media supplemented with sea salt. Colonies could grow at 20-45°C (optimum 28-35°C) and pH 6.0-11.0 (optimum, pH 7.0-9.0), and they could proliferate in the salinity range of 0-6.0 % (w/v) NaCl. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C18 : 1 ω7c 11-methyl, C16 : 0, C16 : 1 ω11c, C16 : 1 ω5c, C17 : 1 ω6c and C18 : 1 ω5c. The respiratory quinone was ubiquinone 10, and the polar lipid profile included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, one unidentified phospholipid and one unidentified aminolipid. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain YIM 152171T within the order Rhodospirillales in a distinct lineage that also included the genus Geminicoccus. The 16S rRNA gene sequence similarities of YIM 152171T to those of Arboricoccus pini, Geminicoccus roseus and Constrictibacter antarcticus were 92.17, 89.25 and 88.91 %, respectively. The assembled draft genome of strain YIM 152171T had 136 contigs with an N50 value of 134704 nt, a total length of 3 001 346 bp and a G+C content of 70.27 mol%. The phylogenetic, phenotypic and chemotaxonomic data showed that strain YIM 152171T (=MCCC 1K08488T=KCTC 92884T) represents a type of novel species and genus for which we propose the name Marinimicrococcus gen. nov., sp. nov.

A novel bi-functional cold-adaptive chitinase from Chitinilyticum aquatile CSC-1 for efficient synthesis of N-acetyl-D-glucosaminidase.

In order to better utilize chitinolytic enzymes to produce high-value N-acetyl-D-glucosamine (GlcNAc) from chitinous waste, there is an urgent need to explore bi-functional chitinases with exceptional properties of temperature, pH and metal tolerance. In this study, we cloned and characterized a novel bi-functional cold-adaptive chitinase called CaChi18A from a newly isolated strain, Chitinilyticum aquatile CSC-1, in Bama longevity village of Guangxi Province, China. The activity of CaChi18A at 50 °C was 4.07 U/mg. However, it exhibited significant catalytic activity even at 5 °C. Its truncated variant CaChi18A_ΔChBDs, containing only catalytic domain, demonstrated significant activity levels, exceeding 40 %, over a temperature range of 5-60 °C and a pH range of 3 to 10. It was noteworthy that it displayed tolerance towards most metal ions at a final concentration of 0.1 mM, including Fe3+ and Cu2+ ions, retaining 122.52 ± 0.17 % and 116.42 ± 1.52 % activity, respectively. Additionally, it exhibited favorable tolerance towards organic solvents with the exception of formic acid. Interestedly, CaChi18A and CaChi18A_ΔChBDs had a low Km value towards colloidal chitin (CC), 0.94 mg mL-1 and 2.13 mg mL-1, respectively. Both enzymes exhibited chitobiosidase and N-acetyl-D-glucosaminidase activities, producing GlcNAc as the primary product when hydrolyzing CC. The high activities across a broader temperature and pH range, strong environmental adaptability, and hydrolytic properties of CaChi18A_ΔChBDs suggested that it could be a promising candidate for GlcNAc production.

Streptomyces koelreuteriae sp. nov., isolated from the rhizosphere soil of Koelreuteria paniculata and healthy leaves of Xanthium sibiricum.

Two actinomycete strains, designated MG62T and CRLD-Y-1, were isolated from rhizosphere soil of Koelreuteria paniculata and healthy leaves of Xanthium sibiricum, respectively, in Hunan province, PR China. They could produce abundant aerial mycelia that generated rod-shaped spores with spiny surfaces. Morphological features of the two strains are typical of the genus Streptomyces. Strains MG62T and CRLD-Y-1 exhibited 99.93 % 16S rRNA gene sequence similarity. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between them were 99.99 and 100 %, respectively, suggesting that they belonged to the same species. 16S rRNA gene sequences analysis revealed that the two strains belonged to the genus Streptomyces and showed highest similarities to Streptomyces violarus NBRC 13104T (99.07-99.29 %) and Streptomyces arenae ISP 5293T (99.21-99.35 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains MG62T and CRLD-Y-1 were closely related to S. violarus NBRC 13104T and S. arenae ISP 5293T. However, the ANI, dDDH and multilocus sequence analysis evolutionary distance values between the two strains and their relatives provide a robust basis upon which to verify strains MG62T and CRLD-Y-1 as representing a novel species. Moreover, a comprehensive comparison of phenotypic and chemotaxonomic characteristics further confirmed that the two strains were distinct from their relatives. Based on all these data above, strains MG62T and CRLD-Y-1 should represent a novel Streptomyces species, for which the name Streptomyces koelreuteriae sp. nov. is proposed. The type strain is MG62T (=JCM 34747T=MCCC 1K06175T).

Comparative Transcriptome Analysis Reveals the Molecular Mechanisms of Acetic Acid Reduction by Adding NaHSO3 in Actinobacillus succinogenes GXAS137.

Acetic acid (AC) is a major by-product from fermentation processes for producing succinic acid (SA) using Actinobacillus succinogenes. Previous experiments have demonstrated that sodium bisulfate (NaHSO3) can significantly decrease AC production by A. succinogenes GXAS137 during SA fermentation. However, the mechanism of AC reduction is poorly understood. In this study, the transcriptional profiles of the strain were compared through Illumina RNA-seq to identify differentially expressed genes (DEGs). A total of 210 DEGs were identified by expression analysis: 83 and 127 genes up-regulated and down-regulated, respectively, in response to NaHSO3 treatment. The functional annotation analysis of DEGs showed that the genes were mainly involved in carbohydrates, inorganic ions, amino acid transport, metabolism, and energy production and conversion. The mechanisms of AC reduction might be related to two aspects: (i) the lipoic acid synthesis pathway (LipA, LipB) was significantly down-regulated, which blocked the pathway catalyzed by pyruvate dehydrogenase complex to synthesize acetyl-coenzyme A (acetyl-CoA) from pyruvate; (ii) the expression level of the gene encoding bifunctional acetaldehyde-alcohol dehydrogenase was significantly up-regulated, and this effect facilitated the synthesis of ethanol from acetyl-CoA. However, the reaction of NaHSO3 with the intermediate metabolite acetaldehyde blocked the production of ethanol and consumed acetyl-CoA, thereby decreasing AC production. Thus, our study provides new insights into the molecular mechanism of AC decreased underlying the treatment of NaHSO3 and will deepen the understanding of the complex regulatory mechanisms of A. succinogenes.

Streptomyces fuscus sp. nov., a brown-black pigment producing actinomycete isolated from dry mudflat sand.

A novel Gram-stain-positive, aerobic actinobacterial strain, designated GXMU-J15T, was isolated from dry mudflat sand. A polyphasic approach was employed for its taxonomic characterization. The strain developed extensively branched yellowish white to light yellow substrate mycelia and white aerial mycelia, and produced smooth cylindrical spores in a loose straight spore chain on International Streptomyces Project 2-7 agar media. Strain GXMU-J15T grew at 20-50 °C (optimum, 35 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-8 % (w/v) NaCl. Analysis of 16S rRNA gene sequences indicated that strain GXMU-J15T represents a member of the genus Streptomyces. Strain GXMU-J15T showed the highest 16S rRNA gene sequence similarity to Streptomyces lusitanus CGMCC 4.1745T (99.1 %) and Streptomyces thermocarboxydus CGMCC 4.1883T (98.8 %). Phylogenetic tree analysis based on multilocus sequence analysis (MLSA) and whole genome sequence construction revealed that strain GXMU-J15T was most closely related to Streptomyces cupreus PSKA01T, Streptomyces cinnabarinus DSM 40467T and Streptomyces davaonensis JCM 4913T. The MLSA and genome-to-genome distances between strain GXMU-J15T and its relatives were 0.0418, 0.0443 and 0.0485 and 0.1237, 0.1188 and 0.1179, respectively. The results of orthologous average nucleotide identity and digital DNA-DNA hybridization analysis corroborated the results of the MLSA and whole genome sequence evolution analysis, indicating that the novel isolate represents a distinct species of the genus Streptomyces. The whole-cell sugars of strain GXMU-J15T were xylose, glucose and galactose. The characteristic diamino acid in the cell-wall hydrolysate was ll-diaminopimelic acid. The lipids contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylglycerides, phosphatidylcholine, two phospholipids of an unknown structure containing glucosamine, one unknown phospholipid and two unknown lipids. The major cellular fatty acid components were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The main respiratory quinone types were MK-9(H6) and MK-9(H8). The whole genome size of strain GXMU-J15T was 8.68 Mbp, with 71.23 mol% G+C content. Genomic analysis indicated that strain GXMU-J15T has the potential to synthesize polyketides, terpenes and a series of important antibiotics besides the gene cluster for melanin synthesis. Based on these genotypic and phenotypic data, strain GXMU-J15T is proposed to represent a new species of the genus Streptomyces named Streptomyces fuscus sp. nov. The type strain is GXMU-J15T (=MCCC 1K08211T=JCM 35917T).

Senkyunolide B exhibits broad-spectrum antifungal activity against plant and human pathogenic fungi via inhibiting spore germination and destroying the mature biofilm.

BACKGROUND: Aspergillus infection seriously jeopardizes the health and safety of life of immunocompromised patients. The emergences of antifungal resistance highlight a demand to find new effective antifungal drugs. Angelica sinensis is a medicine-food herb and phthalides are its characteristic components. A few of the phthalides have been reported to display satisfactory antifungal activities against plant pathogenic fungi. However, the structure-activity relationships and antifungal action mechanism of phthalides remain to be further explored and elucidated. RESULTS: The antifungal activities of five natural phthalides and four artificial analogs were investigated, and their structure-activity relationships were preliminarily elucidated in the current study. The benzene ring moiety played an essential role in their antifungal activities; the oxygen-containing substituents on the benzene ring obviously impacted their activities, the free hydroxyl was favorable to the activity. Typical phthalide senkyunolide B (SENB) exhibited broad antifungal activities against human and plant pathogenic fungi, especially, Aspergillus fumigatus. SENB affected the spore germination and hyphae growth of Aspergillus fumigatus via down-regulating phosphatidylinositol-PKC-calcineurin axis and the expression of ENG genes. Moreover, SENB disturbed the oxidation-reduction process in Aspergillus fumigatus to destroy the mature biofilms. In vivo experiments indicated SENB significantly prolonged survival and decreased fungal burden in mouse model of invasive pulmonary aspergillosis. CONCLUSIONS: Phthalides could be considered as the valuable leads for the development of antifungal drug to cure plant and human disease. © 2023 Society of Chemical Industry.

Caffeine Synthesis and Its Mechanism and Application by Microbial Degradation, A Review.

Caffeine is a metabolite derived from purine nucleotides, typically accounting for 2-5% of the dry weight of tea and 1-2% of the dry weight of coffee. In the tea and coffee plants, the main synthesis pathway of caffeine is a four-step sequence consisting of three methylation reactions and one nucleosidase reaction using xanthine as a precursor. In bacteria, caffeine degradation occurs mainly through the pathways of N-demethylation and C-8 oxidation. However, a study fully and systematically summarizing the metabolism and application of caffeine in microorganisms has not been established elsewhere. In the present study, we provide a review of the biosynthesis, microbial degradation, gene expression, and application of caffeine microbial degradation. The present review aims to further elaborate the mechanism of caffeine metabolism by microorganisms and explore the development prospects in this field.

Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae).

Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution; thus, new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified. To find new toxins, whole genome sequencing of the novel B. thuringiensis strain Bt S3076-1 was performed, and ten predicted toxic genes were identified in this study, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS‒PAGE analysis showed that the major proteins at the spore maturation stage were approximately 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while active proteins after trypsin digestion (approximately 70 kDa and 40 kDa) exhibited LC50 values of 149.64 µg/g and 441.47 µg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed that the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide an experimental reference for further research on the insecticidal activity, toxicity spectrum and synergism of these toxins in Bt S3076-1.

Streptomyces beihaiensis sp. nov., a chitin-degrading actinobacterium, isolated from shrimp pond soil.

A Gram-stain-positive actinobacterium, designated strain GXMU-J5T, was isolated from a sample of shrimp pond soil collected in Tieshangang Saltern, Beihai, PR China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. The organism formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. Phylogenetic analysis of 16S rRNA gene sequences showed that strain GXMU-J5T was most related to Streptomyces kunmingensis DSM 41681T (similarity 97.74 %) and Streptomyces endophyticus YIM 65594T (similarity 96.80 %). However, the values of digital DNA-DNA hybridization, average nucleotide identity and evolutionary distance of multilocus sequence analysis between strain GXMU-J5T and its closest relatives indicated that it represented a distinct species. Strain GXMU-J5T contained ll-diaminopimelic acid and the major whole-cell hydrolysates were xylose and galactose. The predominant menaquinones of strain GXMU-J5T were revealed as MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides and phospholipids of unknown structure containing glucosamine. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. The whole genome size of strain GXMU-J5T was 6.79 Mbp with a 71.39 mol% G+C content. Genomic analysis indicated that strain GXMU-J5T had the potential to degrade chitin. On the basis of these genotypic and phenotypic data, it is supported that strain GXMU-J5T represents a novel species of the genus Streptomyces, for which the name Streptomyces beihaiensis sp. nov. is proposed. The type strain is strain GXMU-J5T (=MCCC 1K08064T=JCM 35629T).

Identification and Surveys of Promoting Plant Growth VOCs from Biocontrol Bacteria Paenibacillus peoriae GXUN15128.

The role of microbial volatile organic compounds (MVOCs) in promoting plant growth has received much attention. We isolated Paenibacillus peoriae from mangrove rhizosphere soil, which can produce VOCs to promote the growth of Arabidopsis thaliana seedlings, increase the aboveground biomass of A. thaliana, and increase the number of lateral roots of A. thaliana. The effects of different inoculation amounts and different media on the composition of MVOCs were studied by solid-phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) and headspace sampler/GC-MS. We found that the growth medium influences the function and composition of MVOCs. To survey the growth-promoting functions, the transcriptome of the receptor A. thaliana was then determined. We also verified the inhibitory effect of the soluble compounds produced by P. peoriae on the growth of 10 pathogenic fungi. The ability of P. peoriae to produce volatile and soluble compounds to promote plant growth and disease resistance has shown great potential for application in the sustainability of agricultural production. IMPORTANCE Microbial volatile organic compounds (MVOCs) have great potential as "gas fertilizers" for agricultural applications, and it is a promising research direction for the utilization of microbial resources. This study is part of the field of interactions between microorganisms and plants. To study the function and application of microorganisms from the perspective of VOCs is helpful to break the bottleneck of traditional microbial application. At present, the study of MVOCs is lacking; there is a lack of functional strains, especially with plant-protective functions and nonpathogenic application value. The significance of this study is that it provides Paenibacillus peoriae, which produces VOCs with plant growth-promoting effects and broad-spectrum antifungal activity against plant-pathogenic fungi. Our study provides a more comprehensive, new VOC component analysis method and explains how MVOCs promote plant growth through transcriptome analysis. This will greatly increase our understanding of MVOC applications as a model for other MVOC research.

Reduced acetic acid formation using NaHSO3 as a steering agent by Actinobacillus succinogenes GXAS137.

The high production of acetic acid (AC) as a by-product leads to difficult separation and purification of succinic acid (SA) and increases production costs in SA fermentation by Actinobacillus succinogenes. NaHSO3 as a steering agent was used to reduce AC production. Herein, the optimum fermentation conditions were achieved by single-factor and orthogonal tests as follows: glucose 60 g/L; MgCO3 60 g/L; NaHSO3 0.15% (w/v); and NaHSO3 addition time, 8 h after inoculation. After optimization, the SA and AC contents were 44.42 and 5.73 g/L. The SA improved by 100.72%, the AC decreased by 21.18% compared with the unfermented. The acetate kinase activity decreased by 14.36% and acetyl-CoA content improved by 97.55% in the group of NaHSO3 addition compared with control check (CK). The mechanism of NaHSO3 is formation acetaldehyde-sodium bisulfite compound and reduction the activity of acetate kinase. These findings indicated a new way of using NaHSO3 as a steering agent to reduce AC generation and may help promote the development of SA industrial production.

TonB systems are required for Aeromonas hydrophila motility by controlling the secretion of flagellin.

The TonB system is required for the active transport of iron compounds across the outer membrane in Gram-negative bacteria. Our previous data indicated that three TonB systems act coordinately to contribute to the motility of Aeromonas hydrophila NJ-35. In this study, we found that flagellum biogenesis was defective in the ΔtonB123 mutant. Subcellular localization indicated that the flagellin subunits FlaA and FlaB were trapped in the cytoplasm of ΔtonB123 mutant with reduced molecular mass. Overexpression of FlaA or FlaB in the ΔtonB123 mutant was unable to restore the secretion of flagellin subunits. Further investigation demonstrated that flagellins in the ΔtonB123 mutant showed a weak affinity for the flagellin-specific chaperone FliS, which is necessary for the export of flagellins. Deglycosylation analysis indicated that flagellins in the cytoplasm of the ΔtonB123 mutant were almost nonglycosylated. Our data suggested that disruption of tonB123 impairs the formation of flagella by inhibiting flagellin glycosylation and decreasing the binding affinity of flagellin for the chaperone FliS. Taken together, our findings indicate a new role of the TonB system in flagellar biogenesis in A. hydrophila.

Acinetobacter faecalis Sp. Nov., Isolated from Elephant Faeces.

A Gram-negative coccobacillus, YIM 103518T, isolated from wild elephant feces in Xishuangbanna, Yunnan Province, West China, was characterized and identified using a polyphasic taxonomic approach. The strain was strictly aerobic, non-motile, catalase-positive and oxidase-negative, colonies were round, convex, smooth, and pale yellow. The strain growth at 4-40 ℃ (optimum, 28 ℃), pH 6.0-10.0 (optimum, pH 7.0) and 0-4% NaCl (optimum, 0%) in culture medium YIM 38. The major fatty acids of strain YIM 103518T were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The predominant ubiquinone was Q-9. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and phospholipids. The 16S rRNA gene sequence showed moderate level of similarity with Acinetobacter portensis AC 877T (98.7%), Acinetobacter sichuanensis CCTCC AB 2018118T (97.1%), and Acinetobacter cumulans CCTCC AB 2018119T (97.1%). The G+C content of the genomic DNA was 36.5 mol%. Strain YIM 103518T showed an average nucleotide identity value of 86.6%, 77.3% and 78.5%, a digital DNA-DNA hybridizations value of 31.2%, 21.9% and 23.0% with the type strain of A. portensis, A. sichuanensis and A. cumulans based on draft genome sequences, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain YIM 103518T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter faecalis sp. nov. is proposed. The type strain is YIM 103518T (=CCTCC AB 2019201T = NBRC 114057T).