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A Seed-Endophytic Bacillus safensis Strain With Antimicrobial Activity Has Genes for Novel Bacteriocin-Like Antimicrobial Peptides.

ABSTRACT: Bacteriocins are a highly diverse group of antimicrobial peptides that have been identified in a wide range of commensal and probiotic organisms, especially those resident in host microbiomes. Rising antibiotic resistance have fueled renewed research into new drug scaffolds such as antimicrobial peptides for use in therapeutics. In this investigation, we examined mung bean seeds for endophytes possessing activity against human and plant pathogens. We isolated a novel strain of Bacillus safensis, from the contents of surface-sterilized mung bean seed, which we termed B. safensis C3. Genome sequencing of C3 identified three distinct biosynthetic systems that produce bacteriocin-based peptides. C3 exhibited antibacterial activity against Escherichia coli, Xanthomonas axonopodis, and Pseudomonas syringae. Robust antimicrobial activity of B. safensis C3 was observed when C3 was co-cultured with Bacillus subtilis. Using the cell-free supernatant of C3 and cation exchange chromatography, we enriched a product that retained antimicrobial activity against B. subtilis. The peptide was found to be approximately 3.3 kDa in size by mass spectrometry, and resistant to proteolysis by Carboxypeptidase Y and Endoproteinase GluC, suggesting that it is a modified variant of an AS-48 like bacteriocin. Our findings open new avenues into further development of novel bacteriocin-based scaffolds for therapeutic development, as well as further investigations into how our discoveries of bacteriocin-producing plant commensal microorganisms may have the potential for an immediate impact on the safety of food supplies.

SUBMITTER: Romero-Severson J

PROVIDER: S-EPMC8503640 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hydrocarbon-degrading and plant-growth-promoting bacterial endophytes have proven useful for facilitating the phytoremediation of petroleum-contaminated soils with high salinity. In this study, we identified Bacillus safensis strain ZY16 as an endophytic bacterium that can degrade hydrocarbons, produce biosurfactants, tolerate salt, and promote plant growth. The strain was isolated from the root of Chloris virgata Sw., a halotolerant plant collected from the Yellow River Delta. ZY16 survived in Luria-Bertani (LB) broth with 0-16% (w/v) sodium chloride (NaCl) and grew well in LB broth supplemented with 0-8% NaCl, indicating its high salt tolerance. The endophytic strain ZY16 effectively degraded C12-C32 n-alkanes of diesel oil effectively, as well as common polycyclic aromatic hydrocarbons under hypersaline conditions. For example, in mineral salts (MS) liquid medium supplemented with 6% NaCl, ZY16 degraded n-undecane, n-hexadecane, n-octacosane, naphthalene, phenanthrene, and pyrene, with degradation percentages of 94.5, 98.2, 64.8, 72.1, 59.4, and 27.6%, respectively. In addition, ZY16 produced biosurfactant, as confirmed by the oil spreading technique, surface tension detection, and emulsification of para-xylene and paraffin. The biosurfactant production ability of ZY16 under hypersaline conditions was also determined. Moreover, ZY16 showed plant-growth-promoting attributes, such as siderophore and indole-3-acetic acid production, as well as phosphate solubilization. To assess the enhanced phytoremediation of saline soils polluted by hydrocarbons and the plant-growth-promotion ability of ZY16, a pot trial with and without inoculation of the endophyte was designed and performed. Inoculated and non-inoculated plantlets of C. virgata Sw. were grown in oil-polluted saline soil, with oil and salt contents of 10462 mg/kg and 0.51%, respectively. After 120 days of growth, significant enhancement of both the aerial and underground biomass of ZY16-inoculated plants was observed. The soil total petroleum hydrocarbon degradation percentage (a metric of phytoremediation) after incubation with ZY16 was 63.2%, representing an elevation of 25.7% over phytoremediation without ZY16 inoculation. Our study should promote the application of endophytic B. safensis ZY16 in phytoremediation by extending our understanding of the mutualistic interactions between endophytes and their host plants.

Project description:The mechanism of bacterial adaption to manganese-polluted environments was explored using 50 manganese-tolerant strains of bacteria isolated from soil of the largest manganese mine in China. Efficiency of manganese removal by the isolated strains was investigated using atomic absorption spectrophotometry. Bacillus safensis strain ST7 was the most effective manganese-oxidizing bacteria among the tested isolates, achieving up to 82% removal at a Mn(II) concentration of 2,200 mg/L. Bacteria-mediated manganese oxide precipitates and high motility were observed, and the growth of strain ST7 was inhibited while its biofilm formation was promoted by the presence of Mn(II). In addition, strain ST7 could grow in the presence of high concentrations of Al(III), Cr(VI), and Fe(III). Genome-wide analysis of the gene expression profile of strain ST7 using the RNA-seq method revealed that 2,580 genes were differently expressed under Mn(II) exposure, and there were more downregulated genes (n = 2,021) than upregulated genes (n = 559) induced by Mn stress. KAAS analysis indicated that these differently expressed genes were mainly enriched in material metabolisms, cellular processes, organism systems, and genetic and environmental information processing pathways. A total of twenty-six genes from the transcriptome of strain ST7 were involved in lignocellulosic degradation. Furthermore, after 15 genes were knocked out by homologous recombination technology, it was observed that the transporters, multicopper oxidase, and proteins involved in sporulation and flagellogenesis contributed to the removal of Mn(II) in strain ST7. In summary, B. safensis ST7 adapted to Mn exposure by changing its metabolism, upregulating cation transporters, inhibiting sporulation and flagellogenesis, and activating an alternative stress-related sigB pathway. This bacterial strain could potentially be used to restore soil polluted by multiple heavy metals and is a candidate to support the consolidated bioprocessing community.

Project description:ObjectiveTo generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo.MethodsThe pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge.ResultsThe recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets.ConclusionThe generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

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A Seed-Endophytic Bacillus safensis Strain With Antimicrobial Activity Has Genes for Novel Bacteriocin-Like Antimicrobial Peptides.

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