Siderophore Production by Rhizosphere Biological Control Bacteria Brevibacillus brevis GZDF3 of Pinellia ternata and Its Antifungal Effects on Candida albicans.

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32°C, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe3+ and Cu2+ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae.

Onions can be damaged by Fusarium basal rot caused by the soilborne fungus Fusarium oxysporum f. sp. cepae (FOC). Control of this pathogen is challenging since there is limited genetic resistance in onion. The identification of molecules that inhibit this pathogen is needed. Antagonism screening showed Brevibacillus fortis NRS-1210 secreted antifungal compounds into growth medium. The spent growth medium, diluted 1:1, inhibited growth of FOC conidia after seven hours and killed 67-91% of conidia after 11 h. The spent medium also inhibited growth of propagules from F. graminearum, F. proliferatum, F. verticillioides and Galactomyces citri-aurantii. Full strength spent growth medium did not effectively kill FOC conidia and chlamydospores inoculated into a sand cornmeal mixture. In silico analysis of the B. fortis NRS-1210 genome indicated the biosynthetic clusters of several antibiotics. Fractionation of spent medium followed by reverse-phase liquid chromatography with tandem mass spectrometry analysis found that fractions with the most antifungal activity contained a combination of edeines A, B and F and no other recognized antibiotics. 1H NMR signals of the active fraction corresponded to edeine, a pentapeptide with broad spectrum antimicrobial activity which blocks translation in both prokaryotes and eukaryotes. Comparative genomics of Brevibacillus genomes shows edeine producers form a clade which consists of: Brevibacillus brevis, Brevibacillus formosus, 'Brevibacillus antibioticus', Brevibacillus schisleri, Brevibacillus fortis, and Brevibacillus porteri. This observation suggests edeine played an important role in the evolution and speciation of the Brevibacillus genus.

Brevibacillus antibioticus sp. nov., with a broad range of antibacterial activity, isolated from soil in the Nakdong River.

A Gram-stain-positive, aerobic, motile, and rod-shaped bacterial strain designated TGS2-1T was isolated from sediment soil in the Nakdong River, Republic of Korea. The optimal growth of strain TGS2-1T was observed at 28°C and pH 7.0 without NaCl supplementation. Strain TGS2-1T revealed antibiosis against various bacteria, including Staphylococcus aureus KCCM 4051, CCARM 3089 (methicillin resistant strains), Enterococcus faecalis KCCM 11814, Escherichia coli KCTC 2443, Candida albicans KACC 7270, and Filobasidium neoformans KCTC 7902. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that strain TGS2-1T belonged to the genus Brevibacillus and shared 93.8-99.7% sequence similarity with Brevibacillus species. Whole-genome sequencing of strain TGS2-1T revealed a genome size of 6.2 Mbp and DNA G + C content of 47.0 mol%. The TGS2-1T genome shared an average nucleotide identity and digital DNA-DNA hybridization of 74.6-93.3% and 18.6-67.1%, respectively, with six related Brevibacillus genomes. The major fatty acid constituents of strain TGS2-1T were anteiso-C15:0 (62.3%) and anteiso-C17:0 (10.8%). Cells of strain TGS2-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, seven unidentified aminophospholipids, and five unidentified lipids. The isoprenoid quinone detected in the strain was menaquinone-7 (MK-7). Based on data obtained from this polyphasic taxonomic study, strain TGS2-1T represents a novel species belonging to genus Brevibacillus, for which the name B. antibioticus sp. nov. is proposed. The type strain is TGS2-1T (= KCCM 90326T = NBRC 113840T = FBCC-B2501).

Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis.

Addition of MgCl2 to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis. The specific activity of the pullulanase obtained from medium supplemented with MgCl2 was also higher than that obtained in culture medium without added magnesium ions. In this work, the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Circular dichroism assays revealed considerable differences in secondary structure between the active and inactive pullulanase forms. Transmission electron microscopy suggested that magnesium ion addition inhibits the shedding of cell wall protein (HWP) layers from the cell surface. Quantitative real-time PCR showed that magnesium ion addition represses transcription of HWP. Because the pullulanase gene and HWP have identical promoters, pullulanase gene transcription was also inhibited. These results suggest that when pullulanase is expressed slowly, it tends to fold into an active form.

Tea saponin enhanced biodegradation of decabromodiphenyl ether by Brevibacillus brevis.

Decabromodiphenyl ether (BDE209) is a ubiquitous persistent pollutant and has contaminated the environment worldwide. To accelerate BDE209 elimination and reveal the mechanism concerned, the biosurfactant tea saponin enhanced degradation of BDE209 by Brevibacillus brevis was conducted. The results revealed that tea saponin could efficiently increase the solubility of BDE209 in mineral salts medium and improve its biodegradation. The degradation efficiency of 0.5 mg L(-1) BDE209 by 1 g L(-1) biomass with surfactant was up to 55% within 5d. Contact time was a significant factor for BDE209 biodegradation. BDE209 biodegradation was coupled with bioaccumulation, ion release and utilization, and debromination to lower brominated PBDE metabolites. During the biodegradation process, B. brevis metabolically released Na(+), NH4(+), NO2(-) and Cl(-), and utilized the nutrient ions Mg(2+), PO4(3-) and SO4(2-). GC-MS analysis revealed that the structure of BDE209 changed under the action of strain and nonabromodiphenyl ethers (BDE-208, -207 and -206), octabromodiphenyl ethers (BDE-203, -197 and -196) and heptabromodiphenyl ether (BDE-183) were generated by debromination.

Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment.

Ochrobactrum sp. N1, Brevibacillus parabrevis N2, B. parabrevis N3 and B. parabrevis N4 were selected when preparing a mixed bacterial consortium based on the efficiency of crude oil utilization. A crude oil degradation rate of the N-series microbial consortium reached upwards of 79% at a temperature of 25 °C in a 3.0% NaCl solution in the shake flask trial. In the mesocosm experiment, a specially designed device was used to simulate the marine environment. The internal tank size was 1.5 m (L)×0.8 m (W)×0.7 m (H). The microbial growth conditions, nutrient utilization and environmental factors were thoroughly investigated. Over 51.1% of the crude oil was effectively removed from the simulated water body. The escalation process (from flask trials to the mesocosm experiment), which sought to represent removal under conditions more similar to the field, proved the high efficiency of using N-series bacteria in crude oil degradation.