Frédérique Gancel

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.

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l−1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l−1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.

Impact of energy supply and oxygen transfer on selective lipopeptide production by Bacillus subtilis BBG21

The influence of power dissipation and volumetric oxygen transfer coefficient k(L)a on Bacillus subtilis productivity of lipopeptides surfactin and fengycin was studied in shake flasks in view of scaling-up of this fermentation process. The experiments performed with different flask sizes, relative filling volumes, and shaking frequencies confirmed clearly that lipopeptide production changed in function of power dissipation, via interfacial gas-liquid contact surface and oxygen supply. It was demonstrated that k(L)a is the key parameter controlling the productivity and the selectivity of the bioreaction. Varying the oxygen transfer conditions, the synthesis could be oriented to mixed production or to surfactin mono-production. The fraction of surfactin towards total lipopeptides produced and the maximal surfactin production both increased with k(L)a increase (surfactin concentration about 2 g L(-1) at k(L)a=0.04-0.08 s(-1)), while the maximal fengycin production (fengycin concentration about 0.3 g L(-1)) was obtained at moderate oxygen supply (k(L)a=0.01 s(-1)).

Effect of Different Temperature Downshifts on Protein Synthesis by Aeromonas hydrophila

The psychrotrophic bacterium Aeromonas hydrophila 7966 was subjected to cold shocks from 30°C to 20°C, 15°C, 10°C, or 5°C, or were incubated at low temperature to determine its adaptative response. The cell protein patterns analyzed by two-dimensional electrophoresis revealed that only a few proteins were underexpressed, whereas numerous new proteins appeared with the decrease of temperature, and some others were overexpressed. Among them, a few constituted cold shock proteins because they were transiently induced, whereas others belong to the acclimatation family proteins. Two cold shock proteins of 11 kDa were synthesized at low level because they were visualized only after radiolabeling or silver staining. Moreover, under our experimental conditions, no major cold shock protein of a molecular mass similar to that of E. coli (7.4 kDa) could be identified.

Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2xa0days whereas it remained stable for B. subtilis 9943 during the 6xa0days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation.

Occurrence of two superoxide dismutases in Aeromonas hydrophila: molecular cloning and differential expression of the sodA and sodB genes.

Aeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD (iron-containing superoxide dismutase), which distinguishes them from Enterobacteriacea. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21.5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22.5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.

Selective fengycin production in a modified rotating discs bioreactor.

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient kLa were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787xa0mgxa0L−1), but also better selectivity of the bioreaction towards fengycin (up to 88xa0%) and better yields of fengycin per glucose (up to 62.9xa0mgxa0g−1), lipopeptides per glucose (up to 71.5xa0mgxa0g−1), fengycin per biomass (up to 309xa0mgxa0g−1) and lipopeptides per biomass (up to 396xa0mgxa0g−1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30xa0min−1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480xa0mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (Pfen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the Ppps promoter from B. subtilis BBG111 was replaced by promoter Pfen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with Pfen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.