Isolation of Antibacterial Active Components from Bacillus subtilis RN8 and Their Mechanism of Action
Objective:In pursuit of novel antibacterial agents,a comprehensive search for microorganisms with antibacterial properties was conducted using soil samples collected from the rural areas in Lijiang,Yunnan Province.This preliminary research aimed to understand the mechanisms behind the observed antibacterial effects.Methods:A selection of six bacterial species-Escherichia coli,Staphylococcus aureus,Bacillus thuringiensis,Xanthomonas oryzae,Bacillus subtilis and Bacillus cereus-alongside eleven fungal species,including Candida albicans,Fusarium oxysporumf.sp.niveum,Fusarium-oxysporumf.sp.cucumebrium,F.oxysporumf sp.luffae Suzukietkawai,Geotrichum candidum,Bremia lactucea Regel,Phytophthora parasitica var.nicotianae,Phytophthora capsici,Aspergillus niger,Penicillium italicum,and Rhizoctonia solani,were employed as indicator organisms to screen for antagonistic activity.These potential antagonists were initially identified using the tube dilution and disc diffusion methods,followed by assessment of mycelial growth rate inhibition to determine their efficacy against the indicator pathogens.The strains that demonstrated antibacterial activity were subjected to further physiological and biochemical testing,complemented by 16S rDNA sequence analysis for accurate identification.Upon isolating active compounds from the fermentation broths of these strains,liquid chromatography-mass spectrometry(LC-MS/MS)was utilized for the structural analysis of these molecules.The triphenyltetrazolium chloride(TTC)assay was conducted to measure the dehydrogenase activity in the cells of E.coli and C.albicans to assess the impact on cellular metabolism.Additionally,spectrophotometric methods were applied to quantify the leakage of protein,DNA,and potassium ions from the cells,providing insight into the potential damage to cellular structures caused by the active compounds.Furthermore,a metabolomic study was carried out on C.albicans after treatment with the active compound RN,providing a deeper understanding of the compound's influence on the metabolic pathways within the fungal cells.Results:The study findings indicate that the fermentation broth from Bacillus subtilis strain RN8 exhibits broad-spectrum antagonistic activity against a panel of 17 test bacteria.The active substance,speculated to be a cyclic peptide designated as RN,demonstrated a notable inhibitory effect,achieving a 73.41％reduction in bacterial growth at a concentration of 12.5 µg/mL and an EC50 of 4.69 μg/mL.When exposed to the RN8 solution at 50x dilution,both E.coli and C.albicans experienced a significant decrease in cell dehydrogenase activity,as measured by the TTC assay,with reductions of 41.64％and 19.65％,respectively,over a 30-min exposure.Meanwhile,measurable leakage of proteins and DNA from the cells was documented using the Erdogan Eliuz method,with E.coli showing a protein leakage rate of 12.46％and C.albicans at 14.80％,while the rates of DNA leakage were recorded at 1.31％and 1.19％,respectively.Additionally,potassium ion(K+)leakage,as determined by atomic absorption spectrophotometry,was 9.52％for E.coli and 7.75％for C.albicans.Moreover,the analysis using the KEGG database highlighted substantial impacts on several key metabolic pathways,which included but were not limited to pyrimidine metabolism,the biosynthesis of phenylalanine,tyrosine,and tryptophan,as well as the metabolism of niacin,niacinamide,valine,leucine,and isoleucine.This perturbation involved a total of 37 metabolites,such as 2'-deoxycytidine,thymidine,methylmalonic acid,shikimic acid,indole,and various amino acids and nucleotides,amongst others.Conclusion:In summary,RN disrupts the cell membrane integrity of the Candida albicans,leading to leakage of cellular components.This disruption is associated with significant alterations in crucial metabolic pathways,thus highlighting the role of RN as an effective antibacterial agent.
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