New strain Brevibacillus laterosporus TSA31-5 produces both brevicidine and brevibacillin, exhibiting distinct antibacterial modes of action against Gram-negative and Gram-positive bacteria.

The growing prevalence of antibiotic resistance has made it imperative to search for new antimicrobial compounds derived from natural products. In the present study, Brevibacillus laterosporus TSA31-5, isolated from red clay soil, was chosen as the subject for conducting additional antibacterial investigations. The fractions exhibiting the highest antibacterial activity (30% acetonitrile eluent from solid phase extraction) were purified through RP-HPLC. Notably, two compounds (A and B) displayed the most potent antibacterial activity against both Escherichia coli and Staphylococcus aureus. ESI-MS/MS spectroscopy and NMR analysis confirmed that compound A corresponds to brevicidine and compound B to brevibacillin. Particularly, brevicidine displayed notable antibacterial activity against Gram-negative bacteria, with a minimum inhibitory concentration (MIC) range of 1-8 µg/mL. On the other hand, brevibacillin exhibited robust antimicrobial effectiveness against both Gram-positive bacterial strains (MIC range of 2-4 µg/mL) and Gram-negative bacteria (MIC range of 4-64 µg/mL). Scanning electron microscopy analysis and fluorescence assays uncovered distinctive morphological alterations in bacterial cell membranes induced by brevicidine and brevibacillin. These observations imply distinct mechanisms of antibacterial activity exhibited by the peptides. Brevicidine exhibited no hemolysis or cytotoxicity up to 512 µg/mL, comparable to the negative control. This suggests its promising therapeutic potential in treating infectious diseases. Conversely, brevibacillin demonstrated elevated cytotoxicity in in vitro assays. Nonetheless, owing to its noteworthy antimicrobial activity against pathogenic bacteria, brevibacillin could still be explored as a promising antimicrobial agent.

Screening of Carbofuran-Degrading Bacteria Chryseobacterium sp. BSC2-3 and Unveiling the Change in Metabolome during Carbofuran Degradation.

Carbofuran is one of the most commonly used N-methylcarbamate-based pesticides and is excellent for controlling pests; however, carbofuran also causes soil and water pollution. Although various studies have been conducted on the bioremediation of pesticide-contaminated soil, the changes occurring in the metabolome during the bioremediation of carbofuran are not fully understood. In this study, the intracellular and extracellular metabolites of the Chryseobacterium sp. BSC2-3 strain were analysed during carbofuran degradation by using a liquid chromatography-mass spectrometry-based metabolomics approach. We found that the BSC2-3 strain extracellularly transformed carbofuran into 3-hydroxycarbofuran. Intracellular metabolite analysis revealed that carbofuran mainly affected aminobenzoate degradation, ubiquinone and terpenoid-quinone biosynthesis, and arginine and proline metabolism. Carbofuran especially affected the metabolic pathway for the degradation of naphthalene and aminobenzoate. Metabolomics additionally revealed that the strain produces disease resistance inducers and plant growth regulators. We also identified the genes involved in the production of indole-3-acetic acid, which is one of the most active auxins. Overall, we identified the metabolic changes induced in carbofuran-degrading bacteria and the genes predicted to be responsible for the degradation of carbofuran.

Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method.

Brown patch caused by the basidiomycete fungus Rhizoctonia solani is an economically important disease of cool-season turfgrasses. In order to manage the disease, different types of fungicides have been applied, but the negative impact of fungicides on the environment continues to rise. In this study, the beneficial bacteria Bacillus velezensis GH1-13 was characterized as a potential biocontrol agent to manage brown patch disease. The strain GH1-13 strongly inhibited the mycelial growth of turf pathogens including different anastomosis groups of R. solani causing brown patch and large patch. R. solani AG2-2(IIIB) hyphae were morphologically changed, and fungal cell death resulted from exposure to the strain GH1-13. In addition, the compatibility of fungicides with the bacterial strain, and the combined application of fungicide azoxystrobin and the strain in brown patch control on creeping bentgrass indicated that the strain could serve as a biocontrol agent. To develop strain-specific detection method, two unique genes from chromosome and plasmid of GH1-13 were found using pan-genome analysis of 364 Bacillus strains. The unique gene from chromosome was successfully detected using both SYBR Green and TaqMan qPCR methods in bacterial DNA or soil DNA samples. This study suggests that application of GH1-13 offers an environmentally friendly approach via reducing fungicide application rates. Furthermore, the developed pipeline of strain-specific detection method could be a useful tool for detecting and studying the dynamics of specific biocontrol agents.

Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation.

In our previous study, a batch fermentation of recombinant Escherichia coli DH5α cells harboring the fmo gene from Methylophaga aminisulfidivorans MP(T) produced indirubin (5.0mg/L) and indigo (920mg/L) in a 5L fermenter containing tryptophan medium (2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl). In this study, it was found that indirubin production greatly increased when 0.36g/L cysteine was added to the tryptophan medium, although cysteine inhibited the growth of the recombinant E. coli harboring the fmo gene. However, the addition of cysteine did not inhibit the expression level and activity of FMO in the cell. Indigo was synthesized by the dimerization of two 3-hydroxyindole molecules under the non-enzymatic reaction. Cysteine influenced the regioselectivity of FMO and enhanced the synthesis of 2-hydroxyindole instead of 3-hydroxyindole, which might function to increase indirubin production. The optimal culture conditions for indirubin production in tryptophan medium were determined from the response surface methodology analysis: 2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl, 0.36g/L (3mM) cysteine, pH 8.0 at 35°C. Under these conditions, the recombinant E. coli cells were capable of producing 223.6mg/L of indirubin from 2g/L of tryptophan. The intracellular accumulation of the indirubin crystals might stress the cell, which may be a main reason for the poor growth of the recombinant E. coli pBlue 1.7.