The complete genome of Bacillus amyloliquefaciens subsp. plantarum CAU B946 contains a gene cluster for nonribosomal synthesis of iturin A.
ABSTRACT: The genome of the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum CAU B946 was 4.02 Mb in size and harbored 3,823 genes (coding sequences [CDS]). Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial compounds. Remarkably, strain CAU B946 possessed a gene cluster involved in synthesis of iturin A.
Project description:The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum YAU B9601-Y2 was 4.24 Mb in size and harbored 3,991 coding sequences (CDS). Giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, CAU B946 possessed a gene cluster involved in synthesis of mersacidin.
Project description:The rhizobacterium Bacillus amyloliquefaciens subsp. plantarum S499 (S499) is particularly efficient in terms of the production of cyclic lipopeptides, which are responsible for the high level of plant disease protection provided by this strain. Sequencing of the S499 genome has highlighted genetic differences and similarities with the closely related rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42 (FZB42). More specifically, a rare 8008 bp plasmid (pS499) harboring a rap-phr cassette constitutes a major distinctive element between S499 and FZB42. By curing this plasmid, we demonstrated that its presence is crucial for preserving the typical physiology of S499 cells. Indeed, the growth rate and extracellular proteolytic activity were significantly affected in the cured strain (S499 P-). Furthermore, pS499 made a significant contribution to the regulation of cyclic lipopeptide production. Surfactins and fengycins were produced in higher quantities by S499 P-, whereas lower amounts of iturins were detected. In line with the increase in surfactin release, bacterial motility improved after curing, whereas the ability to form biofilm was reduced in vitro. The antagonistic effect against phytopathogenic fungi was also limited for S499 P-, most probably due to the reduction of iturin production. With the exception of this last aspect, S499 P- behavior fell between that of S499 and FZB42, suggesting a role for the plasmid in shaping some of the phenotypic differences observed in the two strains.
Project description:Biosurfactants are secondary metabolites with surface active properties and have wide application in agriculture, industrial and therapeutic products. The present study was aimed to screen bacteria for the production of biosurfactant, its characterization and development of a cost effective media formulation for iturin A production. A total of 100 bacterial isolates were isolated from different rhizosphere soil samples by enrichment culture method and screened for biosurfactant activity. Twenty isolates were selected for further studies based on their biosurfactant activity [emulsification index (EI%), emulsification assay (EA), surface tension (ST) reduction] and antagonistic activity. Among them one potential isolate Bacillus sp. RHNK22 showed good EI% and EA with different hydrocarbons tested in this study. Using biochemical methods and 16S rRNA gene sequence, it was identified as Bacillus amyloliquefaciens. Presence of iturin A in RHNK22 was identified by gene specific primers and confirmed as iturin A by FTIR and HPLC. B. amyloliquefaciens RHNK22 exhibited good surface active properties and antifungal activity against Sclerotium rolfsii and Macrophomina phaseolina. For cost-effective production of iturin A, 16 different agro-industrial wastes were screened as substrates, and Sunflower oil cake (SOC) was favouring high iturin A production. Further, using response surface methodology (RSM) model, there was a 3-fold increase in iturin A production (using SOC 4%, inoculum size 1%, at pH 6.0 and 37 °C temperature in 48 h). This is the first report on using SOC as a substrate for iturin A production.
Project description:Iturin A is an important broad-spectrum antifungal cyclic lipopeptide used as an ideal potential biological control agent. However, its application is limited mainly due to the producer strains' low productivity and the high production costs. Here, a potentially industrial strain Bacillus amyloliquefaciens CX-20 was proved to use low-cost rapeseed meal (RSM) as the sole source of all nutrients except the carbon source for the high productivity of iturin A. A fermentation model was first established to analyse the specific roles of different RSM components on iturin A production. Proteins and minerals in RSM were confirmed to play positive role, whereas fibre had negative effect. And the maximal concentration of iturin A was predicted to be more than 1.64 g l-1 by the established evaluation model. Moreover, submerged fermentation of B. amyloliquefaciens CX-20 demonstrated a strong ability to hydrolyse RSM and release water-soluble nutrients. This fermentation broth, a mixture of Bacillus, iturin A and RSM hydrolysate, could simultaneously combat clubroot disease and promote the growth of Brassica napus. In conclusion, this study provides a promising strategy to achieve full utilization of RSM for the production of a combination of value-added biological control agent and biofertilizer.
Project description:BACKGROUND:Iturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon. RESULTS:In this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l-1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14?+?2H]2+, [C15?+?2H]2+, [C16?+?2H]2+ and [C17?+?2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l-1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l-1 by overexpression of a pleiotropic regulator DegQ. CONCLUSIONS:To our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14-C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.
Project description:Controlling toxigenic Fusarium graminearum (FG) is challenging. A bacterial strain (S76-3, identified as Bacillus amyloliquefaciens) that was isolated from diseased wheat spikes in the field displayed strong antifungal activity against FG. Reverse-phase high performance liquid chromatography and electrospray ionization mass spectrometry analyses revealed that S76-3 produced three classes of cyclic lipopeptides including iturin, plipastatin and surfactin. Each class consisted of several different molecules. The iturin and plipastatin fractions strongly inhibited FG; the surfactin fractions did not. The most abundant compound that had antagonistic activity from the iturin fraction was iturin A (m/z 1043.35); the most abundant active compound from the plipastatin fraction was plipastatin A (m/z 1463.90). These compounds were analyzed with collision-induced dissociation mass spectrometry. The two purified compounds displayed strong fungicidal activity, completely killing conidial spores at the minimal inhibitory concentration range of 50 µg/ml (iturin A) and 100 µg/ml (plipastatin A). Optical and fluorescence microscopy analyses revealed severe morphological changes in conidia and substantial distortions in FG hyphae treated with iturin A or plipastatin A. Iturin A caused leakage and/or inactivation of FG cellular contents and plipastatin A caused vacuolation. Time-lapse imaging of dynamic antagonistic processes illustrated that iturin A caused distortion and conglobation along hyphae and inhibited branch formation and growth, while plipastatin A caused conglobation in young hyphae and branch tips. Transmission electron microscopy analyses demonstrated that the cell walls of conidia and hyphae of iturin A and plipastatin A treated FG had large gaps and that their plasma membranes were severely damaged and separated from cell walls.
Project description:Bacillus amyloliquefaciens strain RHNK22 isolated from groundnut rhizosphere showed direct and indirect plant growth-promoting traits along with biosurfactant activity and reduction in surface tension of water. Biosurfactants were identified as lipopeptides (surfactin, iturin, and fengycin) by molecular and biochemical analysis in our studies.
Project description:The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum strain NAU-B3 is 4,196,170 bp in size and harbors 4,001 genes. Nine giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, NAU_B3 contains a large inversion within the central portion of the genome.
Project description:Benzene, toluene, ethylbenzene and (p-, m- and o-) xylene (BTEX) are classified as main pollutants by several environmental protection agencies. In this study, a non-pathogenic, Gram-positive rod-shape bacterium with an ability to degrade all six BTEX compounds, employed as an individual substrate or as a mixture, was isolated. The bacterial isolate was identified as Bacillus amyloliquefaciens subsp. plantarum strain W1. An overall BTEX biodegradation (as individual substrates) by strain W1 could be ranked as: toluene?>?benzene, ethylbenzene, p-xylene?>?m-xylene?>?o-xylene. When presented in a BTEX mixture, m-xylene and o-xylene biodegradation was slightly improved suggesting an induction effect by other BTEX components. BTEX biodegradation pathways of strain W1 were proposed based on analyses of its metabolic intermediates identified by LC-MS/MS. Detected activity of several putative monooxygenases and dioxygenases suggested the versatility of strain W1. Thus far, this is the first report of biodegradation pathways for all of the six BTEX compounds by a unique bacterium of the genus Bacillus. Moreover, B. amyloliquefaciens subsp. plantarum W1 could be a good candidate for an in situ bioremediation considering its Generally Recognized as Safe (GRAS) status and a possibility to serve as a plant growth-promoting rhizobacterium (PGPR).
Project description:Fusarium head blight (FHB) caused by infection with Fusarium graminearum leads to enormous losses to crop growers, and may contaminate grains with a number of Fusarium mycotoxins that pose serious risks to human and animal health. Antagonistic bacteria that are used to prevent FHB offer attractive alternatives or supplements to synthetic fungicides for controlling FHB without the negative effects of chemical management. Out of 500 bacterial strains isolated from soil, Bacillus amyloliquefaciens JCK-12 showed strong antifungal activity and was considered a potential source for control strategies to reduce FHB. B. amyloliquefaciens JCK-12 produces several cyclic lipopeptides (CLPs) including iturin A, fengycin, and surfactin. Iturin A inhibits spore germination of F. graminearum. Fengycin or surfactin alone did not display any inhibitory activity against spore germination at concentrations less than 30 ?g/ml, but a mixture of iturin A, fengycin, and surfactin showed a remarkable synergistic inhibitory effect on F. graminearum spore germination. The fermentation broth and formulation of B. amyloliquefaciens JCK-12 strain reduced the disease incidence of FHB in wheat. Furthermore, co-application of B. amyloliquefaciens JCK-12 and chemical fungicides resulted in synergistic in vitro antifungal effects and significant disease control efficacy against FHB under greenhouse and field conditions, suggesting that B. amyloliquefaciens JCK-12 has a strong chemosensitizing effect. The synergistic antifungal effect of B. amyloliquefaciens JCK-12 and chemical fungicides in combination may result from the cell wall damage and altered cell membrane permeability in the phytopathogenic fungi caused by the CLP mixtures and subsequent increased sensitivity of F. graminearum to fungicides. In addition, B. amyloliquefaciens JCK-12 showed the potential to reduce trichothecenes mycotoxin production. The results of this study indicate that B. amyloliquefaciens JCK-12 could be used as an available biocontrol agent or as a chemosensitizer to chemical fungicides for controlling FHB disease and as a strategy for preventing the contamination of harvested crops with mycotoxins.