Max Béchet

Mycosubtilin Overproduction by Bacillus subtilis BBG100 Enhances the Organism's Antagonistic and Biocontrol Activities

ABSTRACT A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor.

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188xa0mgxa0l−1 for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47xa0mgxa0l−1 for BB1, 207xa0mgxa0l−1 for BB2, and 393xa0mgxa0l−1 for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.

High-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.

ABSTRACT High-level production (880 mg liter−1) and isolation of the anteiso-C17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.

Temperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633.

Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues with fatty acid moiety varying from C15 to C17 are usually co-produced, with their biological activities increasing with the number of carbons in the fatty acid chain. In the present report, we highlight that growth temperature modulates both the extent of mycosubtilin production and the relative abundance of the different homologues. A 30-fold increase in mycosubtilin production was observed when the temperature was decreased from 37 degrees C to 25 degrees C for both strain ATCC6633 and its derivative BBG100, a constitutive mycosubtilin overproducer. However, no significant difference in either the expression of the mycosubtilin synthetase encoding genes or in the intracellular synthetase concentration could be found, suggesting that the observed phenotype originated from a higher mycosubtilin synthetase turnover at lower temperature. We also point out that lower growth temperature leads to an increased proportion of odd-numbered fatty acid homologues as a consequence of de novo synthesis of C17 anteiso fatty acid following cell adaptation to low temperatures.

Production of a novel mixture of mycosubtilins by mutants of Bacillus subtilis.

Using promoter exchange and gene knock-out strategies, two mutant strains, the so-called BBG116 and BBG125, were constructed from Bacillus subtilis wild-type strain ATCC 6633, a surfactin and mycosubtilin producer. Compared to the parental strain, both mutants overproduced constitutively mycosubtilin, while BBG125 had lost the ability to synthesize surfactin. Surprisingly, BBG125 was found to produce about 2-fold less mycosubtilin than BBG116 despite an expected higher availability of the cytoplasmic precursors and cofactors pool for biosynthesis. Further physiological characterization of BBG125 also highlighted: (i) a strong influence of temperature on mycosubtilin biosynthesis in BBG125 with a maximal productivity observed at 22°C, compared to 15 and 30°C; (ii) substantial changes in fatty acid profiles and thereby in mycosubtilin isoforms, compared to the wild-type strain; and (iii) the presence of five novel mycosubtilin isoforms. The antifungal activities of the new mix were higher than or equal to those of purified isoforms.

Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50xa0% response by the exposed test organisms) was about 100xa0mgxa0L−1 in Microtox assays and 6xa0mgxa0L−1 in Daphnia magna immobilization tests for mycosubtilin and 125 mg L−1 and 25xa0mgxa0L−1 for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100xa0mgxa0L−1 of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50xa0mgxa0L−1) gave the same result on B. lactucae development as 100xa0mgxa0L−1 of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.

The Tunisian oasis ecosystem is a source of antagonistic Bacillus spp. producing diverse antifungal lipopeptides.

The use of microbial products has become a promising alternative approach to controlling plant diseases caused by phytopathogenic fungi. Bacteria isolated from the date palm tree rhizosphere of the Tunisian oasis ecosystem could provide new biocontrol microorganisms adapted to extreme conditions, such as drought, salinity and high temperature. The aim of this study was to screen bacteria isolated from the rhizosphere of the date palm tree for their ability to inhibit phytopathogenic fungi, and to identify molecules responsible for their antifungal activity. Screening for antifungal activity was performed on twenty-eight isolates. Five antagonistic isolates were selected and identified as different species of Bacillus using phenotypical methods and a molecular approach. The five antagonistic Bacillus isolated showed tolerance to abiotic stresses (high temperature, salinity, drought). Their ability to produce lipopeptides was investigated using a combination of two techniques: PCR amplification and MALDI-ToF mass spectrometry. Analyses revealed that the antagonistic isolates produced a high diversity of lipopeptides that belonged to surfactin, fengycin, iturin and kurstakin families. Their antagonistic activity, related to their capacity for producing diverse antifungal lipopeptides and their tolerance to abiotic stresses, highlighted Bacillus strains isolated from the rhizosphere of the date palm tree as potential biocontrol agents for combatting plant diseases in extreme environments.

Production of Bacillus amyloliquefaciens OG and its metabolites in renewable media: valorisation for biodiesel production and p-xylene decontamination

Biosurfactants are important in many areas; however, costs impede large-scale production. This work aimed to develop a global sustainable strategy for the production of biosurfactants by a novel strain of Bacillus amyloliquefaciens. Initially, Bacillus sp. strain 0G was renamed B. amyloliquefaciens subsp. plantarum (syn. Bacillus velezensis) after analysis of the gyrA and gyrB DNA sequences. Growth in modified Landys medium produced 3 main recoverable metabolites: surfactin, fengycin, and acetoin, which promote plant growth. Cultivation was studied in the presence of renewable carbon (as glycerol) and nitrogen (as arginine) sources. While diverse kinetics of acetoin production were observed in different media, similar yields (6-8 g·L-1) were obtained after 72 h of growth. Glycerol increased surfactin-specific production, while arginine increased the yields of surfactin and fengycin and increased biomass significantly. The specific production of fengycin increased ∼10 times, possibly due to a connecting pathway involving arginine and ornithine. Adding value to crude extracts and biomass, both were shown to be useful, respectively, for the removal of p-xylene from contaminated water and for biodiesel production, yielding ∼70 mg·g-1 cells and glycerol, which could be recycled in novel media. This is the first study considering circular bioeconomy to lower the production costs of biosurfactants by valorisation of both microbial cells and their primary and secondary metabolites.