RESUMO
Bacillus sp. has proven to be a goldmine of diverse bioactive lipopeptides, finding wide-range of industrial applications. This review highlights the importance of three major families of lipopeptides (iturin, fengycin, and surfactin) produced by Bacillus sp. and their diverse activities against plant pathogens. This review also emphasizes the role of non-ribosomal peptide synthetases (NRPS) as significant enzymes responsible for synthesizing these lipopeptides, contributing to their peptide diversity. Literature showed that these lipopeptides exhibit potent antifungal activity against various plant pathogens and highlight their specific mechanisms, such as siderophore activity, pore-forming properties, biofilm inhibition, and dislodging activity. The novelty of this review comes from its comprehensive coverage of Bacillus sp. lipopeptides, their production, classification, mechanisms of action, and potential applications in plant protection. It also emphasizes the importance of ongoing research for developing new and enhanced antimicrobial agents. Furthermore, this review article highlights the need for future research to improve the production efficiency of these lipopeptides for commercial applications. It recognizes the potential for these lipopeptides to expand the field of biological pest management for both existing and emerging plant diseases.
Assuntos
Anti-Infecciosos , Bacillus , Bacillus/genética , Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , Plantas/microbiologia , Bacillus subtilisRESUMO
Bacillus subtilis species complex is known as lipopeptide-producer with biotechnological potential for pharmaceutical developments. This study aimed to identify lipopeptides from a bacterial isolate and evaluate their antifungal effects. Here, we isolated and identified a lipopeptide-producing bacterium as a species of Bacillus subtilis complex (strain UL-1). Twenty lipopeptides (six iturins, six fengycins, and eight surfactins) were identified in the crude extract (CE) and fractions (F1, F2, F3, and F4), and the highest content of total lipopeptides was observed in CE and F2. The chemical quantification data corroborate with the hemolytic and antifungal activities that CE and F2 were the most hemolytic and inhibited the fungal growth at lower concentrations against Fusarium spp. In addition, they caused morphological changes such as shortening and/or atypical branching of hyphae and induction of chlamydospore-like structure formation, especially in Fusarium solani. CE was the most effective in inhibiting the biofilm formation and in disrupting the mature biofilm of F. solani reducing the total biomass and the metabolic activity at concentrations ≥ 2 µg/mL. Moreover, CE significantly inhibited the adherence of F. solani conidia on contact lenses and nails as well as disrupted the pre-formed biofilms on nails. CE at 100 mg/kg was nontoxic on Galleria mellonella larvae, and it reduced the fungal burden in larvae previously infected by F. solani. Taken together, the lipopeptides obtained from strain UL-1 demonstrated a potent anti-Fusarium effect inducing morphological alterations and antibiofilm activities. Our data open further studies for the biotechnological application of these lipopeptides as potential antifungal agents. KEY POINTS: ⢠Lipopeptides inhibit Fusarium growth and induce chlamydospore-like structures. ⢠Lipopeptides hamper the adherence of conidia and biofilms of Fusarium solani. ⢠Iturins, fengycins, and surfactins were associated with antifungal effects.
Assuntos
Antifúngicos , Bacillus subtilis , Bacillus subtilis/metabolismo , Antifúngicos/química , Esporos Fúngicos/metabolismo , Biofilmes , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Doenças das Plantas/microbiologiaRESUMO
The relationship between lipopeptide and primary metabolite production by Bacillus spp. in solid-state fermentation (SSF) and submerged fermentation (SmF) was evaluated. Four wild-type strains and one mutant strain (unable to develop biofilm) were assessed in SSF and SmF, using a defined medium and polyurethane foam as inert support for SSF. Strain ATCC 21,332 in SSF presented the highest lipopeptide production. The wild-type strains revealed higher lipopeptide and biomass production and lower synthesis of primary metabolites in SSF than in SmF. However, the mutant strain showed a slightly higher production of primary metabolites in SSF than in SmF. Carbon balance analysis showed that the carbon flux was mainly directed to lipopeptides in SSF, whereas in SmF, it was directed to the production of primary metabolites and the carbon flux to lipopeptides is inversely related to primary metabolites in both types of cultures.
Assuntos
Bacillus , Biomassa , Meios de Cultura , Fermentação , LipopeptídeosRESUMO
Cyclic lipopeptides (CLPs) from Bacillus strains have demonstrated a wide range of bioactivities making them interesting candidates for different applications in the pharmaceutical, food and biotechnological industries. Genome sequencing, together with phylogenetic analysis of the Bacillus sp. P34, isolated from a freshwater fish gut, showed that the bacterial strain belongs to the Bacillus velezensis group. In silico investigation of metabolic gene clusters of nonribosomal peptide synthetases (NRPS) revealed the genetic elements associated with the synthesis of surfactin, fengycin and iturin family component bacillomycin. Further, an assay was conducted to investigate the production of CLPs in the presence of heat inactivated bacterial cultures or fungal spores. Maximum fengycin concentration was observed at 24 h (2300-2700 mg/mL), while maximum iturin amounts were detected at 48 h (250 mg/mL) in the presence of heat-inactivated spores of Aspergillus niger. Heat-inactivated cells of Listeria monocytogenes caused a reduction of both fengycin and iturin amounts. The production of fengycins A and B and the iturin family component bacillomycin L was confirmed by mass spectrometry analyses. This study reinforces the potential of B. velezensis P34 as a valuable strain for biotechnological production of CLPs recognized as important antimicrobial substances.
Assuntos
Bacillus/genética , Bacillus/metabolismo , Lipopeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , Animais , Aspergillus niger , Bacillus/isolamento & purificação , Hibridização Genômica Comparativa , Peixes/microbiologia , Genoma Bacteriano , Listeria monocytogenes , Anotação de Sequência Molecular , Filogenia , Staphylococcus aureus , Sequenciamento Completo do GenomaRESUMO
Bacterial endophytes are well known inhabitants of living plant system and perform important assignments in maintaining plant growth and health. Currently, limited reports are available on the endophytes of pearl millet (Pennisetum glaucum) reflecting antagonistic and plant growth promoting (PGP) attributes. Therefore, the major objectives of current investigation were to identify antagonistic strains of endophytic Bacillus from pearl millet and further illustrate their PGP capabilities. In this study, 19 endophytic Bacillus strains (EPP5, EPP21, EPP30, EPP32, EPP35, EPP42, EPP49, EPP55, EPP62, EPP65, EPP70, EPP71, EPP74, EPP78, EPP83, EPP86, EPP93, EPP100, and EPP102) displaying antagonistic activity towards Rhizoctonia solani (RS), Sclerotium rolfsii (SR), and Fusarium solani (FS) were isolated from different sections (root, leaf, stem, and root) of pearl millet. Phenotypic (shape, colony, gram staining reaction, endospore formation, and motility) and biochemical features (catalase, oxidase, citrate, gelatinase, urease, Voges Proskauer's, methyl red, indole, and nitrate reduction), along with the similarly comparison of 16S rRNA gene sequence with type strains identified eight antagonistic endophyhtes as B. amyloliquefaciens (EPP35, EPP 42, EPP62, and EPP 102), Bacillus subtilis subsp. subtilis (EPP65), and Bacillus cereus (EPP5, EPP71, and EPP74). The production of indole acetic acid and siderophores was varied among the isolated endophytes. Besides displaying enzymatic activities, these isolates varied in solubilizing capabilities of phosphate, potassium, and zinc. The presence of three antimicrobial peptide genes (ituD, bmyC, and srfA) also confirmed their antifungal nature. Further, single treatment of three promising strains (EPP5, EPP62, and EPP65) offered protection ranging from 35.68 to 45.74% under greenhouse conditions. However, microbial consortium (EPP5+ EPP62 + EPP65) provided the highest protection (71.96%) against root rot and wilt infection with significant increase in plant biomass. Overall, the current study indicated that pearl millet plant harbors various species of endophytic Bacillus that possess excellent biocontrol and growth promotion activities.
Assuntos
Antifúngicos/isolamento & purificação , Endófitos/isolamento & purificação , Pennisetum , Doenças das Plantas , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/metabolismo , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/isolamento & purificação , Bacillus amyloliquefaciens/metabolismo , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Bacillus cereus/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/metabolismo , Basidiomycota/efeitos dos fármacos , Agentes de Controle Biológico , Endófitos/genética , Endófitos/metabolismo , Fusarium/efeitos dos fármacos , Genes Bacterianos , Ácidos Indolacéticos/metabolismo , Consórcios Microbianos , Interações Microbianas , Pennisetum/crescimento & desenvolvimento , Pennisetum/microbiologia , Fosfatos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Raízes de Plantas/microbiologia , Potássio/metabolismo , Rhizoctonia/efeitos dos fármacos , Sideróforos/metabolismo , Microbiologia do Solo , Zinco/metabolismoRESUMO
The rapid and accurate quantification of lipopeptide families in biological samples are challenging. We present the development and validation of a method for simultaneous quantification of three families of lipopeptides (iturins, fengycins, and surfactins) and their isoforms, as well as the homologous series. The method was optimized in UPLC-MS for a column temperature at 65 °C, injection volume of 5 µL, and sample temperature of 10 °C. The SIM mode was used for detection and quantification of lipopeptides exhibiting ions [M + H]+ and [M + 2H]2+. Since the maximum mass detection threshold of the equipment is 1250 Da and the fengycins have ions between 1435 and 1505 Da, the ions [M + 2H]2+ were chosen for fengycin identification. The monitored ions were as follows: m/z 1043.5, 1057.5, 1071.5, 718.3, 725.4, 739.4, 732.4, 746.4, 753.4, 1008.6, 1022.6, and 1036.6. The compounds were separated by reverse-phase chromatography using a C18 analytical column in a total time of 19 min. Standard curves were linear with rw 0.99 for all analytes. Intra- and inter-day precision for samples (50, 250, and 750 µg L-1) were within recommended limits. The proposed analytical method was capable of simultaneously quantifying 12 isoforms and homologous series of lipopeptide families in biological samples, thus making it an important industrial tool in the evaluation of lipopeptide production processes. Graphical abstract á .
Assuntos
Bacillus subtilis/metabolismo , Fermentação , Lipopeptídeos/análise , Isoformas de Proteínas/metabolismo , Cromatografia de Fase Reversa/métodos , Meios de Cultura , Limite de Detecção , Espectrometria de Massas/métodos , Padrões de Referência , Reprodutibilidade dos Testes , TemperaturaRESUMO
Bacillus strains isolated from the aquatic environment of the Brazilian Amazon region were tested for their activity against mycotoxigenic fungi. All tested bacteria showed antifungal activity, inhibiting at least 7 indicator fungi. Four Bacillus strains showing promising antifungal results were subsequently evaluated for their activity in reducing mycelial growth rate, sporulation, spore germination percentage, and mycotoxin production. Bacillus sp. P1 and Bacillus sp. P11 had a remarkable antifungal effect on toxigenic fungi. Washed bacterial cell suspension of strains P1 and P11 (107CFU/ml) reduced by >70% the fungal colony diameters, including a complete inhibition of ochratoxin A (OTA) producing Aspergillus spp. Significant reduction of growth rate, sporulation and spore germination were also observed. The bacteria influenced the production of mycotoxins, causing a reduction around 99 and 97% in AFB1 and OTA concentration, respectively. Chromatographic analysis revealed the presence of lipopeptides (iturin A and surfactin isomers) in butanol extracts of cell-free supernatants and cell pellets of strains P1 and P11. Furthermore, antifungal activity of these extracts was confirmed against A. flavus A12 and A. carbonarius ITAL293, producers of AFB1 and OTA, respectively. These bacterial strains could be promising biocontrol agents against toxigenic fungi.
Assuntos
Bacillus/fisiologia , Fungos/fisiologia , Intestinos/microbiologia , Interações Microbianas/fisiologia , Micotoxinas/metabolismo , Animais , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/fisiologia , Bacillus/isolamento & purificação , Brasil , Peixes/microbiologia , Fungos/efeitos dos fármacos , Lipopeptídeos/análise , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Ocratoxinas/metabolismoRESUMO
Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Aspergillus/química , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/biossíntese , Expressão Gênica , Staphylococcus aureus/química , Ativação TranscricionalRESUMO
Background In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. Results The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). Conclusion According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments.
Assuntos
Peptídeos Cíclicos , Redes Neurais de Computação , Algoritmos , Fermentação , Técnicas de Cultura Celular por Lotes , Máquina de Vetores de SuporteRESUMO
Background Optimization of nutrient feeding was developed to improve the growth of Bacillus subtilis in fed batch fermentation to increase the production of jiean-peptide (JAA). A central composite design (CCD) was used to obtain a model describing the relationship between glucose, total nitrogen, and the maximum cell dry weight in the culture broth with fed batch fermentation in a 5 L fermentor. Results The results were analyzed using response surface methodology (RSM), and the optimized values of glucose and total nitrogen concentration were 30.70 g/L and 1.68 g/L in the culture, respectively. The highest cell dry weight was improved to 77.50 g/L in fed batch fermentation, which is 280% higher than the batch fermentation concentration (20.37 g/L). This led to a 44% increase of JAA production in fed batch fermentation as compared to the production of batch fermentation. Conclusion The results of this work improve the present production of JAA and may be adopted for other objective products' production.