Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp.

Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.

Deletion of Rap-phosphatases for quorum sensing control in Bacillus and its effect on surfactin production.

The complex regulatory network in Bacillus, known as quorum sensing, offers many opportunities to modify bacterial gene expression and hence to control bioprocesses. One target regulated by this mechanism is the activity of the PsrfA promoter, which is engaged in the formation of lipopeptide surfactin. It was hypothesised that deletion of rapC, rapF and rapH, encoding for prominent Rap-phosphatases known to affect PsrfA activity, would enhance surfactin production. Therefore, these genes were deleted in a sfp+ derivative of B. subtilis 168 with subsequent evaluation of quantitative data. Up to the maximum product formation of the reference strain B. subtilis KM1016 after 16 h of cultivation, the titers of the rap deletion mutants did not exceed the reference. However, an increase in both product yield per biomass YP/X and specific surfactin productivity qsurfactin was observed, without any considerable effect on the ComX activity. By extending the cultivation time, a 2.7-fold increase in surfactin titer was observed after 24 h for strain CT10 (ΔrapC) and a 2.5-fold increase for CT11 (ΔrapF) compared to the reference strain KM1016. In addition, YP/X was again increased for strains CT10 and CT11, with values of 1.33 g/g and 1.13 g/g, respectively. Interestingly, the effect on surfactin titer in strain CT12 (ΔrapH) was not as distinct, although it achieved the highest promoter activity (PsrfA-lacZ). The data presented support the possibility of involving the quorum sensing system of Bacillus in bioprocess control as shown here on the example of lipopeptide production.

Characterization of Bacillus velezensis UTB96, Demonstrating Improved Lipopeptide Production Compared to the Strain B. velezensis FZB42.

Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Bacillus velezensis UTB96 Is an Antifungal Soil Isolate with a Reduced Genome Size Compared to That of Bacillus velezensis FZB42.

Bacillus velezensis UTB96 was isolated from soil based on its antifungal activity. Whole-genome sequencing of strain UTB96 provided further information about its secondary metabolite gene clusters. Compared to the well-known strain FZB42, UTB96 lacks an IS3 element and a type I restriction endonuclease.