Combined Use of NMR, LC-ESI-MS and Antifungal Tests for Rapid Detection of Bioactive Lipopeptides Produced by Bacillus

The science and technology interact with the art in several ways. Biotechnological coupled with analytical approaches can play an important role in protecting and preserving cultural heritage for future generations. Many microorganisms influenced by environmental conditions are the main responsible for biological contamination in built heritage. Biocides based on chemical compounds have been used to mitigate this problem. Thus, it is vitally important to develop proper remediation actions based on environmentally innocuous alternative. Bacillus specie is emerging as an optimistic alternative for built heritage treatment due to their capacity to produce secondary metabolites with antagonistic activities against many fungal pathogens. Therefore, the intent of this work was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using spectroscopic (NMR) and chromatographic techniques (LC-ESI-MS). The high antifungal activity obtained for Bacillus sp. active compounds produced in this study confirms the great potential to suppress biodeteriogenic fungi growth on historical artworks. Additionally, the proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and could be used as a viable alternative to be employed for screening and production of new green biocides.

Lipopeptides for the Fragment-Based Pharmaceutics Design

This paper describes the synthesis of peptide fragments for use in a new type of combinatorial discovery technology, in which the building blocks are brought together by non-covalent interactions, rather than direct chemical bonding. The building blocks of interest—in this case different amino acids—are converted to amphiphiles by attachment to lipid tails. The amphiphiles, when mixed together in aqueous phase, are designed so that they aggregate spontaneously to form micelles. The building blocks form the headgroups of each of the amphiphiles, and these headgroups cover the surface of the micelle in a dynamic close-packed fluid mosaic array. These building blocks come together so closely that two- or three-dimensional structures are created on the surface of the micelles, and these can be screened in biological assays to find out which combination of building blocks is able to elicit a biological response. Lipopeptides consisting of two residues of lipoamino acid and other amino acids moieties have been designed, synthesized, characterized and the ability of these constructs to form supra-molecular assemblies is demonstrated.

Safe antifungal lipopeptides derived from Bacillus marinus B-9987 against grey mold caused by Botrytis cinerea

Agricultural application studies, including field experiments and acute toxicity tests, were conducted for lipopeptides secreted by marine-derived Bacillus marinus B-9987. Benefiting from commercially available scaled-up lipopeptide purification, the sample of impurities（isolated from target lipopeptides）, raw extracted sample（purity： 9.08%）, partially purified sample（purity： 20.86%）, and highly purified sample（purity： 87.51%） were prepared from B. marinus B-9987 fermentation broth, and used in lab-scale antagonism tests, field experiments, swarming motility tests, and acute toxicity tests. Operations and conditions in field experiments were consistent with the Pesticide-Guidelines for the Field Efficacy Trials（GB/T 17980.28-2000）, and acute toxicity tests were executed according to Toxicological Test Methods of Pesticides for Registration（GB 15670-1995）. In agar diffusion tests in vitro and pot tests in vivo, all lipopeptide samples with different purities significantly inhibited Botrytis cinerea; meanwhile the sample of impurities isolated from target lipopeptides were not effective against B. cinerea. Results of lab-scale tests showed that the target lipopeptides were effective substances against B. cinerea. Thus, partially purified and raw extracted samples were used in field experiments instead of the highly purified sample for cost saving. In the field experiments against rose grey mold, biological control efficacy of 500 mg L–1 lipopeptides reached 67.53%, slightly lower than 74.05% reached by the agrochemical pyrimethanil. However, pyrimethanil severely suppressed B. marinus B-9987, whereas the lipopeptides promoted swarming motility and biocontrol efficacy of Bacillus biomass. Lipopeptides at 87.51% purity were tested for systemic acute toxicity and confirmed as low-toxicity substances. In conclusion, low-toxicity lipopeptides were potential alternatives to agrochemicals, and they also performed good promotion when combined with homologous biological control microorganism. There were 2 breakthroughs in this research：（1） marine-derived bacterial lipopeptides inhibited grey mold caused by B. cinerea in field experiments; and（2） purified bacterial lipopeptides（sample purity： 〉87.51%） were determined to be low-toxicity substances by systemic acute toxicity tests, satisfying the strict requirement of pesticide registration in China（required purity： 〉85%）. This study provides support for using extracellularBacillus-derived lipopeptides commercially similar to Bacillus-based biological control agents.

Comparative Study of Disease Suppression on Various Host Plants by <i>Bacillus</i>Cyclic Lipopeptides

In biological controls using Bacillus spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between cyclic lipopeptides and host plants to induce disease resistance. In this study, we aimed to clarify the specificity to induce disease resistance among cyclic lipopeptides on various host plants. Our data clearly showed both cyclic lipopeptides conferred disease suppression on most of host plants, but at different range of cyclic lipopeptide concentration. Our findings contribute to understanding the complex on the specificity of cyclic lipopeptide derived induced disease resistance.

Lysohexaenetides A and B,linear lipopeptides from Lysobacter sp.DSM 3655 identified by heterologous expression in Streptomyces

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.

Translocation of subunit PPSE in plipastatin synthase and synthesis of novel lipopeptides

Nonribosomal peptide synthase(NRPS)is a unique molecular assembly mechanism with high hybridity.Its recombination is conducive to the development of novel lipopeptides.However,there are few reports on NRPS subunit recombination of plipastatin at present.In this paper,plipastatin synthase was modified by the forward movement of subunit PPSE and the replacement of the communication-mediating(COM)domain.The results showed that ppsABE,a new assembly line,could synthesize novel lipopeptides such as cycle pentapeptide(C16-18β-OHFA-E-O-cyclo(Y-T-I),and its antimicrobial activity against Rhizopus stolonifer and Staphylococcus aureus was better than that of plipastatin.However,the reactivity of ppsABCE disappeared,but the substitution of COM^(D)_(ppsC)/COM^(A)_(ppsD) or COM^(D)_(ppsD)/COM^(A)_(ppsE) for COM^(D)_(ppsC)/COM^(A)_(ppsE) could restore its activity and conduct the biosynthesis of linear hexapeptide(C16-17β-OHFA-E-O-Y-T-E-A/V)and heptapeptide(C17-18β-OHFA-E-O-Y-T-E-A-I).Collectively,these findings indicated that the COM donor domain at the C-terminus of PPSB could communicate with the COM acceptor domain at the N-terminus of PPSE and that the compatible COM domain is an important tool for communication between nonpartner subunits.Moreover,the integrity and selective compatibility of the COM acceptor domain of subunit PPSE are essential to promote the interaction between PPSE and other subunits.This work further complemented the rules of NRPS subunit recombination and provided a theoretical basis for the development of novel high-efficiency lipopeptides.

Lipopeptides in promoting signals at surface/interface of micelles:Their roles in repairing cellular and nuclear damages

Lipopeptides are recognized as a structurally diverse group of functional biopeptides.They possess multiple biological,biotechnological and therapeutic applications and are able to affect cell membrane integrity and permeability.It is widely accepted that suppressing the interaction of redooxidation is mainly aimed by inserting functional biopeptides on form alpha-helical peptides or on form antibody.Such types of structured biomaterials have great potential in promoting signal transductions to the most organelles having geometrical and spacial structures similar to endogenous liposome forms such as lysosomes,ribosomes,mitochondria and platelets..Thus,inserting radioactive trace-elements along with biopeptides is necessary in tracing the trajectory of those radioactive micelles that are destined in targeting further complicated disease and growth factor propagation.These techniques are important in determining structure-symmetry and asymmetry leading to meso-spherical crystal-packing modes or to copolymer vesiculization.The main objective of this review is to highlight and elucidate the potential role of lipopeptides in promoting signals at surface/interface of micelles along with their functionalities in repairing cellular and nuclear damages.Moreover,state-of-the-art scientific knowledge is reviewed regarding bioconjugation and structuring charged-mono-layer and/or bi-layer phospholipidic membranes in adherent to signal-biopeptides using some advanced techniques such as ultra-sound probe based polydispersity of radioactive trace-elements assisted microdialysis as micro-separating techniques in between those miscible and immiscible compounds.

Bacteriostatic effect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

Bacillus subtilis N-2 which was isolated from natto,produced lipopeptides using soybean meal as a substrate.This work aimed to purify,identify,and determine the antibacterial mechanism of lipopeptides produced by B.subtilis N-2.The fermented product obtained by solid-state fermentation was subjected to water extraction,acid precipitation,and methanol extraction.Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC-MS/MS.Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the p-amino fatty acid chain from C14 to C17 as well as the amino acid positions.A new lipopeptide(m/z 1070.3)was identified and its structure was different from the previously reported lipopeptides.The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida,a common pathogen in decaying fish,by changing membrane permeability.These results suggest that the lipopeptides from B.subtilis N-2 could be used as a biocontrol agent in aquaculture.

Pseudomonas Cyclic Lipopeptide Medpeptin:Biosynthesis and Modulation of Plant Immunity

The multifunctional secondary metabolites known as cyclic lipopeptides(CLPs),which are produced by a large variety of bacteria,have become a key category of plant immunity elicitors.Pseudomonas-CLPs(PsCLPs)are extremely diverse in structure and biological activity.However,an understanding of CLP-plant structure–function interactions currently remains elusive.Here,we identify medpeptin,a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids.Medpeptin is synthesized by a non-ribosomal peptide synthase(NRPS)gene cluster and regulated by a quorum-sensing system.Further research indicates that medpeptin does not exhibit antimicrobial activity;instead,it induces plant cell death immunity and confers resistance to bacterial infection.Comparative transcriptome analysis and virus-induced gene silencing(VIGS)reveal a set of immune signaling candidates involved in medpeptin perception.Silencing of a cell-wall leucine-rich repeat extensin protein(NbLRX3)or a receptor-like protein kinase(NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana.Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.

Transcriptomics reveals substance biosynthesis and transport on membranes of Listeria monocytogenes affected by antimicrobial lipopeptide brevilaterin B

Listeria monocytogenes is a worrisome food-borne pathogen threatening global food safety.Our previous study proved that lipopeptide brevilaterin B showed efficient antibacterial activity against L.monocytogenes by interacting with the cell membrane.This research further explored the antibacterial mechanism of brevilaterin B against L.monocytogenes at the sub-minimum inhibition concentration via transcriptomic analysis.Brevilaterin B induced growth inhibition rather than direct membrane lysis in L.monocytogenes at the minimum inhibitory concentration.Transcriptomic analysis showed 1779 difference expressed genes,including 895 up-regulated and 884 down-regulated genes.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that brevilaterin B influenced multiple pathways of L.monocytogenes,including peptidoglycan biosynthesis,membrane transport(ATP-binding cassette transports,ion transport),cellular metabolism(amino acid and lipid metabolism),ATP synthesis,and activation of the stress response(quorum sensing and bacterial chemotaxis).In conclusion,brevilaterin B affects gene expression related to biosynthesis,transport and stress response pathways on the membrane of L.monocytogenes.The present work provides the first transcriptomic assessment of the antibacterial mechanism of lipopeptide brevilaterin B at the gene level.

Metabolites Composition of Bacillus subtilis HussainT-AMU Determined by LC-MS and Their Effect on Fusarium Dry Rot of Potato Seed Tuber

Fusarium dry rot is considered to be the most critical soilborne and postharvest disease that damages potato tubers worldwide when they are stored for a long time.This study was performed to demonstrate the effect of crude extract,culture filtrate,and cell suspension obtained from the bacterium Bacillus subtilis Hussain T-AMU,on the net house and field.From oil-contaminated soil,through the serial dilution method,biosurfactant bacteria were isolated on nutrient agar medium.To isolate and screen the prospective biosurfactant strain,various biosurfactant screening methods were used.Standard protocols were carried out for morphological,molecular,and chemical characterization.The disease incidences were significantly decreased under net house and field conditions after treatment with biosurfactant extract(20%,70%),culture filtrate(23%,49%),and bacterial cell suspension(46%,27%),respectively.These results indicate a significant effect of B.subtilis HussainT-AMU against Fusarium oxysporum.The characterization of the partially purified extract by gas-chromatography indicated the existence of a high amount of methyl ester fatty acids.The quantitative analysis determined the presence of lipopeptide-surfactin,fengycin,and iturin in the biosurfactant extract.Scanning electron microscopy revealed that morphological variation was revealed in the structure of stomata size in leaves and in the mycelia of the pathogen that was investigated.This promising biocontrol agent can be a plausible option for further research and can also be used for the production of green biomolecules at an additional industrial level.

Biosurfactant-producing Ability of Highly Efficient Petroleum-degrading Bacterium BS-8

After incubation for 6 -30 h, with the rapid increase of bacterial cell number, surface tension of bacterium BS-8 was reduced sharply from 63.2 mN/m to 39.4 mN/m. The production of biosurfactants by BS-8 was growth-dependent. Using glucose as the carbon source, bacterium BS-8 was incubated. Based on centrifugation, precipitation and chromogenie reaction of the culture solution, results indicated that the biosurfactants belonged to lipopeptides. The yield of biosurfaetants isolated and purified from the culture solution was 0.58 g/L, and the critical micelle concentration （CMC） was 90 mg,/L. Under conditions of pH 4 -9, tem- perature 20 -70 ℃, NaCl concentration 1% -6%, biosurfactants predueed by BS-8 exhibited the highest stability.

Isolation and characterization of halophilic Bacillus sp.BS3 able to produce pharmacologically important biosurfactants

Objective:To characterize the pharmacological importance of biosurfactants isolated from halophilic Bacillus sp BS3.Methods:Halophilic Bacillus sp.BS3 was isolated from solar salt works,identified by 16S rRNA sequencing and was used for screening their biosurfactant production.Characters of the biosurfactant and their anticancer activity were analyzed and performed in mammary epithelial carcinoma cell at different concentrations.Results:The biosurfactant were characterized by TLC,FTIR and GC-MS analysis and identified as lipopeptide type.GC-MS analysis revealed that,the biosurfactant had various compounds including 13Docosenamide.(Z);Mannosamine,9- and N,N,N',N'-tetramethyl.Surprisingly the antiviral activity was found against shrimp white spot syndrome virus(WSSV) by suppressing the viral replication and significantly raised shrimp survival(P<0.01).Anticancer activity performed in the mammary epithelial carcinoma cell at different concentrations of biosurfactants,among the various concentrations of biosurfactants such as 0.000 25,0.002 5,0.025,0.25 and 2.5 μ g,the 0.25 μ g concentration suppressed the cells significantly(P<0.05) to 24.8%.Conclusions:Based on the findings,the present study concluded that,there is a possibility to develop eco-friendly antimicrobial and anticancer drugs from the extremophilic origin.

C_(17)-fengycin B,produced by deep-sea-derived Bacillus subtilis,possessing a strong antifungal activity against Fusarium solani

Root rot disease caused by Fusarium solani is the most devastating disease of the tomato and legume crops in China.The metabolites of Bacillus species can inhibit many fungal diseases.In this study,the metabolites of deep-sea-derived bacterium Bacillus subtilis 2 H11 can significantly inhibit the growth of F.solani.The metabolite C_(17)-fengycin B,one of the cyclic lipopeptides,was identified by the combination of silica column chromatography,high-performance liquid chromatography(HPLC),high-energy collision induced dissociation mass spectrometry(HCD-MS)and tandem mass spectrometry(HCD-MS/MS).The results of scanning electron microscopy(SEM)and transmission electron microscopy(TEM)showed that C_(17)-fengycin B could destroy the structure of the hyphae and spores of F.solani.The antifungal activities of C_(17)-fengycin B against F.solani were tested at concentrations ranging from 0.05 mg/mL to 0.20 mg/mL.The results indicated that C_(17)-fengycin B inhibited the growth of F.solani with antifungal index of 89.80%at 0.20 mg/mL,and the antifungal activity of C_(17)-fengycin B was further verified by the pot experiment.In addition,the cytotoxicity experiment showed that C_(17)-fengycin B had good biocompatibility and was a potential candidate for the development of biocontrol pesticide in the future.

Functional characterization of a biosurfactant-producing thermo-tolerant bacteria isolated from an oil reservoir

A Bacillus subtilis strain JA-1 isolated from an oil reservoir was studied. This strain is capable of growth and producing biosurfactant at a temperature of 60 ℃. In nutrient medium it produced biosurfactant which reduced the surface tension from 68.2 mN/m to 28.3 mN/m, with the critical micelle concentration （CMC） of 48 mg/L. The measured surface tension indicated that the biosurfactant possessed stable surface activity at high temperature and a specific range of pH and salt concentrations. The results of thin layer chromatography （TLC） together with FT-IR showed that the metabolic product of strain JA-1 is a lipopeptide biosurfactant. The ability to growth at high temperature and to produce biosurfactant makes strain JA-1 promising for enhanced oil recovery.

Derringer desirability and kinetic plot LC-column comparison approach for MS-compatible lipopeptide analysis

Lipopeptides are currently re-emerging as an interesting subgroup in the peptide research field, having historical applications as antibacterial and antifungal agents and new potential applications as antiviral, antitumor, immune-modulating and cell-penetrating compounds. However, due to their specific structure, chromatographic analysis often requires special buffer systems or the use of trifluoroacetic acid, limiting mass spectrometry detection. Therefore, we used a traditional aqueous/acetonitrile based gradient system, containing 0.1% （m/v） formic acid, to separate four pharmaceutically relevant lipopeptides （polymyxin B1, caspofungin, daptomycin and gramicidin A1）, which were selected based upon hierarchical cluster analysis （HCA） and principal component analysis （PCA）.In total, the performance of four different C18 columns, including one UPLC column, were evaluated using two parallel approaches. First, a Derringer desirability function was used, whereby six single and multiple chromatographic response values were rescaled into one overall D-value per column. Using this approach, the YMC Pack Pro C18 column was ranked as the best column for general MS-compatible lipopeptide separation. Secondly, the kinetic plot approach was used to compare the different columns at different flow rate ranges. As the optimal kinetic column performance is obtained at its maximal pressure, the length elongation factor λ（Pmax/Pexp） was used to transform the obtained experimental data （retention times and peak capacities） and construct kinetic performance limit （KPL） curves, allowing a direct visual and unbiased comparison of the selected columns, whereby the YMC Triart C18 UPLC and ACE C18 columns performed as best. Finally, differences in column performance and the （dis）advantages of both approaches are discussed.

Lipopeptide Antibiotics Produced by the Engineered Strain Bacillus subtilis GEB3 and Detection of Its Bioactivity

MALDI-TOF-MS technology was used for identification of lipopeptide antibiotics producedby GEB3 strain, a derivative of Bacillus subtilis 168 which was transformed by lpaB3gene. The result showed GEB3 only produced lipopeptide antibiotic surfactin. The analysisby LC-MS demonstrated that GEB3 produced standard surfactin isoforms with side chainlengths of 13,14 and 15 carbon atoms. The bioactivity detection of surfactin indicatedthat the surfactin produced by GEB3 had inhibition effect on plant pathogens Rhizoctoniasolani and Pyricularia oryzae.

Optimized Biosurfactant Production by <i>Pseudomonas aeruginosa</i>Strain CGA1 Using Agro-Industrial Waste as Sole Carbon Source

Biosurfactants are biomolecules produced by microorganisms, which possess several advantages over their chemical counterparts. Production can be cost-effective if renewable wastes are utilized as substrates. In this study, optimization of biosurfactant production by Pseudomonas aeruginosa strain CGA1 was carried out using response surface methodology. The conventional “One factor at a time” method of optimization was initially adopted to ascertain the impact of different renewable wastes on biosurfactant production. Four independent variables were tested: carbon and nitrogen concentration, medium volume, and inoculum size. Biosurfactant production was based on the emulsification index measurement. Results indicated that the preferred carbon source by the isolate was sugar cane molasses. A 2.31-fold increase in biosurfactant yield and emulsification index of 96.3% ± 0.75% under optimized cultural conditions of 20 g/L of molasses, 5 g/L of sodium nitrate, 1.93 ml inoculum size and 60 ml medium volume in 250 ml conical flask were obtained. The regression coefficient (R2) value of 84.15% implied adequate fitness of the model. The surface tension of distilled water was reduced from 72.1 mN/m to 35.0 ± 0.0 mN/m, and critical micelle concentration was attained at 60 mg·L-1. FTIR and GC-MS analysis indicated that the biosurfactant was a lipopeptide having characteristic lipid and peptide peak values. This study proves that the sole use of agro-industrial wastes for the production of biosurfactant is very efficient, and ensures the economic feasibility of biosurfactant production.

Isolation and characterization of biosurfactant producing bacteria from groundnut oil cake dumping site for the control of foodborne pathogens

Infection and intoxication are two common types of foodborne illnesses throughout the world.The aim of the present work was to isolate and characterize biosurfactant producing bacteria fromgroundnut oil cake dumping sites and evaluate their biocontrol effects against foodborne pathogens.Bacteria were isolated by enrichment culture technique and preliminary screening method.Biosurfactant activity evaluation was carried out by oil displacement,drop collapse test,lipase activity,hemolytic activity,emulsification index and emulsification assay methods.16s rRNA sequence analysis was used for the isolate identification.Crude biosurfactant was extracted by acid precipitation method and characterized using FTIR(Fourier Transform Infrared Spectroscopy).Antibacterial activity was investigated using disc diffusion method.CMCand surface reduction were analyzed by DuNouy tensiometer.Top 10 strains were selected for biosurfactant activity assessment from the total 30 isolates.In addition,16s rRNA sequence identified that the potential isolate was Bacillus pseudomycoides OR 1.Then,FTIR result of the extracted biosurfactant established the extract as a lipopeptide based on the absorption peaks at 3500 to 3200 cm^-1 and 2963 to 2854.68 cm^-1,respectively.50μg/mL of lipopeptide showed the highest antibacterial activity.Critical Micelle Concentration(CMC)of the lipopeptide was 60 mg/L and it reduced the surface tension of water from 71.6 to 31.6 mN/m.Hence,this study widens the scope to employ the bacterial lipopeptide surfactant as a promising biocontrol agent against foodborne pathogens.

Integrated and dual-responsive lipopeptide nanovector with parallel effect to tumor and micro-environment regulation by efficient gene and drug co-delivery

The integrated lipopeptide(RVA)/gene complexes are fabricated with bi-directional regulation on tumor cells and micro-environment.After self-assembling and target coating modification,the poly(γ-glutamic acid)(γ-PGA)/RVA nano-vectors can sequentially respond to pH&redox stimuli,and guarantee efficient therapeutic gene delivery and control release of all-trans retinoic acid.The design provides a facile but promising strategy to treat refractory cancers.