Composition and activity of antifungal lipopeptides produced by Bacillus spp. in daqu fermentation.

Daqu is a solid-state fermentation and saccharification starter for the Chinese liquor baijou. During the daqu stage, amylolytic and proteolytic enzymes are produced by Bacillus and fungi. Bacillus spp. also produce lipopeptides with a broad spectrum of antimicrobial activities but direct evidence for their impact on community assembly in daqu is lacking. This study aimed to study the interaction between Bacillus spp. and fungi in daqu models. The antifungal activity of surfactin, fengycin, and iturin A was initially assessed in vitro. Iturin A displayed the strongest antifungal activity (MIC = 10-50 mg/L). In situ antifungal activity of B. amyloliquefaciens and B. velezensis against molds was observed in a simple daqu model inoculated with single strains of Bacillus species. Formation of lipopeptides in situ was supported by quantification of mRNA encoding for enzymes for surfactin, fengycin, and iturin A biosynthesis. In situ antifungal activity of Bacillus species was also observed in a complex daqu model that was inoculated with 8 bacterial or fungal strains plus one of the three strains of Bacillus. A relationship of lipopeptides to in situ antifungal activity was further supported by detection of the lipopeptides by liquid chromatography coupled to mass spectrometry. Both results indicated that B velezensis FUA2155 had higher antifungal activity in the daqu model, and was the only strain that produced multiple iturin A congeners in situ. Taken together, this study provides evidence that production of lipopeptides by Bacillus species in daqu may impact community assembly and hence product quality.

Identification of Antifungal Compounds from Avocado Rhizobacteria (Bacillus spp.) against Fusarium spp., by a Bioassay-Guided Fractionation Approach.

Antimicrobial compounds produced by bacteria have been increasingly acknowledged as an important resource for the control of phytopathogens. We used a bioassay-guided fractionation approach to identify antifungal metabolites produced by two avocado rhizobacteria (INECOL-4742 and INECOL-5927), both members of the Bacillus subtilis/B. amyloliquefaciens species complex, against Fusarium solani and F. kuroshium, causal agent of Fusarium dieback in avocado and other hosts. The butanol (BuOH) organic extract from INECOL-4742 (B1-Bu) exhibited the highest percentage of inhibition (PI) against F. solani (78.76 %), also inhibiting F. kuroshium by up to 44.30 %. Primary fractions, Bu-F3, Bu-F12 and Bu-F15, obtained by silica gel open column chromatography, exhibited the highest PI against F. solani (28.57 % to 33.50 %) and F. kuroshium (38.78 % to 45.00 %). The presence of cyclic lipopeptides from the iturin, surfactin and fengycin families in B1-Bu extracts and primary fractions was determined by UPLC-ESI-HRMS. The Confocal Laser Microscopy analysis revealed deformations in the hyphae of F. kuroshium exposed to extracts, primary fractions and C-13 surfactin chemical standard. These results emphasize the potential of natural products from Bacillus for the control of the emerging phytopathogenic fungus F. kuroshium.

Maize Root Exudates Recruit Bacillus amyloliquefaciens OR2-30 to Inhibit Fusarium graminearum Infection.

Bacillus spp. can exert plant growth-promoting effects and biocontrol effects after effective colonization, and bacterial chemotaxis toward plant root exudates is the initial step to colonize. Under biotic stress, plants are able to alter their root exudates to attract or avoid different types of microbes. Hence, Bacillus chemotaxis toward root exudates after pathogen infection is crucial for exerting their beneficial effects. In this study, the Bacillus amyloliquefaciens OR2-30 strain, which exhibited greater chemotaxis ability toward maize root exudates after Fusarium graminearum infection, was screened from 156 rhizosphere microorganisms. The infected maize root exudates were further confirmed to improve the swarming and biofilm formation ability of the OR2-30 strain. Chemotaxis, swarming, and biofilm formation ability were able to influence bacterial colonization. Indeed, the the OR2-30 strain displayed more effective colonization ability in the maize rhizosphere after F. graminearum inoculation. Moreover, lipopeptides produced by OR2-30 were identified as iturins and responsible for suppressing F. graminearum growth. Further study showed that lipopeptides suppressed the growth of F. graminearum by inhibiting conidia formation and germination, inducing reactive oxygen species production and causing cell death in mycelium. Eventually, the OR2-30 strain increased maize resistance against F. graminearum. These results suggested that maize root exudates could recruit B. amyloliquefacines OR2-30 after F. graminearum infection, and that OR2-30 then suppresses the F. graminearum by producing lipopeptides, such as iturins, to protect maize.

Bacillus siamensis CNE6- a multifaceted plant growth promoting endophyte of Cicer arietinum L. having broad spectrum antifungal activities and host colonizing potential.

Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 µg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60-80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC-MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168.

BACKGROUND: Bacillus subtilis is a well-established host for a variety of bioproduction processes, with much interest focused on the production of biosurfactants such as the cyclic lipopeptide surfactin. Surfactin production is tightly intertwined with quorum sensing and regulatory cell differentiation processes. As previous studies have shown, a non-sporulating B. subtilis strain 3NA encoding a functional sfp locus but mutations in the spo0A and abrB loci, called JABs32, exhibits noticeably increased surfactin production capabilities. In this work, the impacts of introducing JABs32 mutations in the genes spo0A, abrB and abh from 3NA into strain KM1016, a surfactin-forming derivative of B. subtilis 168, was investigated. This study aims to show these mutations are responsible for the surfactin producing performance of strain JABs32 in fed-batch bioreactor cultivations. RESULTS: Single and double mutant strains of B. subtilis KM1016 were constructed encoding gene deletions of spo0A, abrB and homologous abh. Furthermore, an elongated abrB version, called abrB*, as described for JABs32 was integrated. Single and combinatory mutant strains were analysed in respect of growth behaviour, native PsrfA promoter expression and surfactin production. Deletion of spo0A led to increased growth rates with lowered surfactin titers, while deletion or elongation of abrB resulted in lowered growth rates and high surfactin yields, compared to KM1016. The double mutant strains B. subtilis KM1036 and KM1020 encoding Δspo0A abrB* and Δspo0A ΔabrB were compared to reference strain JABs32, with KM1036 exhibiting similar production parameters and impeded cell growth and surfactin production for KM1020. Bioreactor fed-batch cultivations comparing a Δspo0A abrB* mutant of KM1016, KM681, with JABs32 showed a decrease of 32% in surfactin concentration. CONCLUSIONS: The genetic differences of B. subtilis KM1016 and JABs32 give rise to new and improved fermentation methods through high cell density processes. Deletion of the spo0A locus was shown to be the reason for higher biomass concentrations. Only in combination with an elongation of abrB was this strain able to reach high surfactin titers of 18.27 g L-1 in fed-batch cultivations. This work shows, that a B. subtilis strain can be turned into a high cell density surfactin production strain by introduction of two mutations.

Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications.

BACKGROUND: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. RESULTS: In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. CONCLUSION: The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin.

Detection of simultaneous production of kurstakin, fengycin and surfactin lipopeptides in Bacillus mojavensis using a novel gel-based method and MALDI-TOF spectrometry.

Bacterial lipopeptides have become a research focus of many studies owing to their industrial and pharmaceutical importance. Although such studies focused on researching purification procedures and qualitative analysis, much remains to be explored and developed to improve the current methods. To enable thorough studies of lipopeptides, this paper describes a new method for purification and characterization of in-gel anionic lipopeptides. Specifically, lipopeptides attributed to the anti-staphylococcal activity of Bacillus mojavensis HF were separated using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and subsequently characterized using mass spectrometry. Lipopeptide band obtained by gel electrophoresis was first visualized using three different staining methods. Next, the lipopeptide isomers were efficiently recovered from the gel band and structural characterization of the extracted lipopeptides was carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS analysis revealed that Bacillus mojavensis HF produced three types of lipopeptides including surfactin, fengycin, and kurstakin. 14 clusters of ion peaks were identified as fengycin A with fatty acid of C15-C17, fengycin B (C16, C17), surfactin (C13-C16), and kurstakin (C9-C12). Moreover, tandem mass spectrometric analysis (MS/MS) revealed the sequences of fengycin A and surfactin. In this study, we identified a high variety and number of surfactin and fengycin isomers, which previous reports lacked. To the best of our knowledge, we are the first to report the presence of kurstakin in Bacillus mojavensis species. Finally, we demonstrated that our gel-based study of lipopeptides allowed for a precise and reproducible investigation of these molecules.

Characterization of extracellular and cell bound biosurfactants produced by Aneurinibacillus aneurinilyticus isolated from commercial corn steep liquor.

The presence of biosurfactants produced by a Bacillus strain in corn steep liquor (CSL), a wastewater stream of the corn milling process, has been recently discovered. However, the species responsible for their production has not been identified at the moment. Therefore, in this work, the Bacillus strain isolated from CSL, with capacity to produce biosurfactants, was subjected to amplification and sequence analysis of the 16S rRNA, being identified as Aneurinibacillus aneurinilyticus. This strain has been proved to be endospore forming and thermophile, what would explain its presence in the commercial CSL. It was observed that the strain under evaluation has the ability to produce both cell-bound and extracellular biosurfactant extracts, which were characterized in this work. The electrospray ionization mass spectrometry (ESI) analysis of the biosurfactant extracts revealed that the extracellular biosurfactant produced by Aneurinibacillus aneurinilyticus is composed by a mixture of lipopeptides, containing C16 and C18 fatty acids and amino acids, including valine, phenylalanine, proline, cysteine, histidine, aspartic acid/asparagine, alanine, glycine, leucine/isoleucine, with biomarkers between 1025-458 m/z. Conversely, the cell-bound biosurfactant extract produced by Aneurinibacillus aneurinilyticus was composed by the cyclic decapeptide gramicidin S, with a characteristic peak at 571 m/z, and lipopeptides with characteristic peaks between 1034-705 m/z, containing alanine, glycine, cysteine, serine, proline, aspartic acid/asparagine, similarly to the amino acid sequence of the extracellular biosurfactant extract.

Chemical Tagging of Protein Lipoylation.

Protein lipoylation is a post-translational modification of emerging importance in both prokaryotes and eukaryotes. However, labeling and large-scale profiling of protein lipoylation remain challenging. Here, we report the development of iLCL (iodoacetamide-assisted lipoate-cyclooctyne ligation), a chemoselective reaction that enables chemical tagging of protein lipoylation. We demonstrate that the cyclic disulfide of lipoamide but not linear disulfides can selectively react with iodoacetamide to produce sulfenic acid, which can be conjugated with cyclooctyne probes. iLCL enables tagging of lipoylated proteins for gel-based detection and cellular imaging. Furthermore, we apply iLCL for proteomic profiling of lipoylated proteins in both bacteria and mammalian cells. In addition to all of the eight known lipoylated proteins, we identified seven candidates for novel lipoylated proteins. The iLCL strategy should facilitate uncovering the biological function of protein lipoylation.

15N Stable Isotope Labeling and Comparative Metabolomics Facilitates Genome Mining in Cultured Cyanobacteria.

As genome mining becomes a more widely used approach to identify bacterial natural products, the challenge of matching biosynthetic gene clusters to their cognate secondary metabolites has become more apparent. Bioinformatic platforms such as AntiSMASH have made great progress in predicting chemical structures from genetic information, however the predicted structures are often incomplete. This complicates identifying the predicted compounds by mass spectrometry. Secondary metabolites produced by cyanobacteria represent a unique opportunity for bridging this gap. Cultured cyanobacteria incorporate inorganic nitrogen provided in chemically defined media into all nitrogen-containing secondary metabolites. Thus, stable isotope labeling with 15N labeled nitrate and subsequent comparative metabolomics can be used to match biosynthetic gene clusters to their cognate compounds in cell extracts. Analysis of the sequenced genome of Nostoc sp. UIC 10630 identified six biosynthetic gene clusters predicted to encode the production of a secondary metabolite with at least one nitrogen atom. Comparative metabolomic analysis of the 15N labeled and unlabeled cell extracts revealed four nitrogen containing compounds that contained the same number of nitrogen atoms as were predicted in the biosynthetic gene clusters. Two of the four compounds were new secondary metabolites, and their structures were elucidated by NMR, HRESIMS, and MS/MS.

Effect-directed screening of Bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography.

Bacillus species produce a wide array of biologically active metabolites, including nonribosomaly synthesized lipopeptides (LPs). The high-performance thin-layer chromatography (HPTLC) technique hyphenated with different bioassays and mass spectrometry was demonstrated as a valuable tool for effect-directed analysis of iturins, surfactins, fengycins and kurstakins homologues from complex mixtures of LPs. As proof of this straightforward strategy, the found surfactin and iturin A homologues were characterized and compared with reference substances. This study considered two different extraction methods for LPs produced by five Bacillus strains. The ethyl acetate extraction (Ex-1), and the acidic precipitation followed by methanol extraction (Ex-2) were investigated. Diverse enzyme inhibitions and antimicrobial potentials of LPs were analyzed, and in parallel, high-resolution mass spectra (HRMS) were online recorded from the HPTLC zones of interest. No antimicrobial effect against Gram-positive B. subtilis was evident for iturin, whereas a response was detected for surfactin. The nonpolar kurstakin compounds showed a pronounced B. subtilis antimicrobial activity in Ex-1 of almost all strains, whereas the fengycin homologues were detected in Ex-2 of SS-10.7 and SS-27.2. Iturin had also no activity against Gram-negative Aliivibrio fischeri, while again surfactin showed an enhancing luminescent activity. Contrary, kurstakin compounds caused a decrease in the luminescence in Ex-1 of all strains, particularly for SS-13.1. Both, iturin and surfactin showed a strong acetylcholinesterase (AChE) and α-glucosidase inhibition, but surfactin caused a much stronger inhibition. This was evident in all bacterial strains, except for SS-13.1 in Ex-1 and for SS-38.4 in Ex-2. Although, iturin and surfactin exhibited no DPPH˙ scavenging activity, Ex-1 of all strains contained more intense DPPH˙ scavenging compounds compared to Ex-2, and surfactin methyl esters showed a pronounced DPPH˙ activity, particularly in SS-12.6 in Ex-1. This study pointed to active metabolites of strains that can be used as therapeutics and biocontrol agents with beneficial effects on human health. The straightforward HPTLC profiling served as an excellent bioanalytical tool to control the formed bioactive metabolites. As the fermentation process is very sensitive to external influences, it could be a helpful control tool for standardization of the biotechnological processing.

Bioactivity of Fermented Green Coffee Bean Extract Containing High Chlorogenic Acid and Surfactin.

Chlorogenic acid (CGA) is a major component of green coffee beans. Surfactin, a cyclic lipopeptide, is produced and secreted by Bacillus subtilis strains. In this study, bioactivities of fermented green coffee bean extract (FGCBE) and the individual compounds, CGA and surfactin. were compared in HepG2 cells. The concentration of surfactin and CGA in the FGCBE and non-fermented green coffee bean extract (NFGCBE) were determined to be 9.2 and 7.33 and 0.72 and 0.53 mg·mL-1, respectively. The FGCBE contained about 20% and 26% more CGA and surfactin than the NFGCBE. Although CGA and surfactin exhibited cytotoxicity at concentrations more than 100 and 20 µg respectively, the FGCBE 50 containing CGA (460 µg·mL-1) and surfactin (720 µg·mL-1) effectively prevented cell death by oxidative stress and also strongly activated the proliferation of cells incubated with under 50 µM H2O2. The CGA and surfactin in FGCBE were 9.2 and 72 times higher than the CGA and surfactin compounds (50 and 10 µg·mL-1). The relative proliferation of the FGCBE-treated cells also was 3.3 and 8.8 times higher than the CGA and surfactin compounds treated the oxidative stressed cells with 50 µM H2O2. These results suggest that the single compounds such as CGA and surfactin generally have cytotoxicity at low concentration of them but FGCBE contained them acted as strong antioxidants, activators of cell proliferation, inhibitors of cell apoptosis. Various bioactive compounds in fermented coffee bean also seem to help cell proliferation and decreasing of cytotoxicity by CGA and surfactin in coffee bean.

Identification of fusaricidins from the antifungal microbial strain Paenibacillus sp. MS2379 using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Paenibacillus sp. MS2379 is a highly efficient microbial strain producing fusaricidins, a class of lipopeptides that have demonstrated strong antifungal activities against a broad array of fungal pathogens. An integrated approach combining chromatographic fractionation, UHPLC-QTOF-MS analysis, and NMR spectroscopic interpretation was employed to characterize antifungal metabolites produced by this microbial strain, resulting in the identification of 48 fusaricidins including 30 cyclic and 18 open-chain species. In this regard, UHPLC-QTOF-MS played a vital role in determining structures of 28 new fusaricidins through peptide fragment analysis. The structural determination of the new fusaricidins by the high-resolution mass spectrometry was validated by follow-up isolation and NMR spectroscopic analysis of representative compounds. It is worth noting that novel fusaricidins with amino acid residues of serine and γ-aminobutyric acid were identified, which is of great biosynthetic significance for this biologically important class of compounds. The present study again illustrates the power of UHPLC-QTOF-MS for structural identification of lipopeptides, and the structural diversity of the identified fusaricidins makes this microbial strain unique as a potential biocontrol agent.

Fast Equivalency Estimation of Unknown Enzyme Inhibitors in Situ the Effect-Directed Fingerprint, Shown for Bacillus Lipopeptide Extracts.

The hyphenation of high-performance thin-layer chromatography (HPTLC) with enzyme inhibition assays followed by high-resolution mass spectrometry (HRMS) represents a targeted profiling of complex natural samples required in the development of new natural pharmaceuticals, functional foods, and cosmetics. This direct combination of a chromatogram with an enzymatic assay substantially extents the understanding of inhibitor properties in vitro. For the first time, a straightforward workflow was established for estimating the equivalency of unknown inhibitors directly in the autogram. Exemplarily, lipopeptides produced as secondary metabolites by five different Bacillus strains were analyzed by HPTLC hyphenated with the tyrosinase and acetylcholinesterase (AChE) assays. Lipopeptides that showed an inhibition were characterized by HPTLC-HRMS. Among the many reports about the biological properties of lipopeptides, their enzyme inhibitory properties are new. The most intense inhibitors were identified as surfactin and iturin A according to reference substances and exact masses. Three further inhibitors were supposedly assigned as fengycin, iturin C, and surfactin methyl ester according to their exact masses. The inhibitory activities of surfactin and iturin A were quantitatively compared with kojic acid and piperine, as references for common natural inhibitors. Their equivalently calculated tyrosinase inhibition showed that 1 µg kojic acid was equal to 1.8 µg and 3.2 µg of iturin A and surfactin standards, respectively; regarding to AChE inhibition, 1 µg piperine was equal to 1.7 µg and 0.6 µg of iturin A and surfactin, respectively. Further unknown enzyme inhibitors found in the sample were exemplarily calculated as surfactin, iturin A, kojic acid, and piperine equivalents to estimate their importance.

Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22.

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 ᅟ.

Biosurfactant-facilitated leaching of metals from spent hydrodesulphurization catalyst.

AIMS: To investigate the capabilities of different types of biosurfactants (rhamnolipids, lipopeptides, sophorolipids) to remove metals and carbon from the hazardous spent hydrodesulphurization (HDS) catalyst generated by petroleum refineries. METHODS AND RESULTS: Biosurfactants were prepared and used to treat spent HDS catalyst. Metal and carbon contents were analysed and compared with those from no-biosurfactant control treatments. All biosurfactant treatments increased carbon loss percentage from the spent HDS catalyst. The lipopeptide treatment LI, containing 17·34 mg ml-1 of crude biosurfactants, caused the highest carbon loss percentage (44·5%). Rhamnolipids were, in general, better than sophorolipids and lipopeptides as metal-removing agents. The metal content decreased as the concentration of rhamnolipids decreased. The R5 treatment, which contained 0·4 mg l-1 of crude rhamnolipids, caused the highest reduction in metal content. Molybdenum, nickle and vanadium contents were reduced by 90, 30 and 70% respectively. CONCLUSIONS: Biosurfactants might have potential application for metals and coke removal from spent HDS catalysts. The bioleaching capability depends on the type and concentration of the biosurfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, after further in-depth investigations, might lead to the development of an eco-friendly and economic technology to treat or even regenerate the environmentally hazardous spent HDS catalysts, which are generated in huge amounts by the petroleum refineries.

Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity.

Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.

Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy.

AIMS: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens. METHODS AND RESULTS: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed. CONCLUSION: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta.

Detergent-assisted sample preparation for MALDI-MS: Investigation of octylglucoside and docecylmaltoside for matrix crystallization, on-plate digestion, and trypsin activity.

We show an easy and fast method for improved detection of lipophilic peptides with MALDI-MS utilizing the nonionic detergents n-octylglucoside and n-dodecylmaltoside (laurylmaltoside). Investigations comprised on-plate digestion of proteins with trypsin, detergent effects on the protease trypsin, and the changes in MALDI matrix crystallization. Investigations also exhibited a higher tryptic activity in trypsin activity assay of 139% when using laurylmaltoside as supplement. Crystallization changed toward a more homogeneous crystal distribution and especially trypsinized insulin spectra recorded with MALDI-MS showed improved detectability of lipophilic peptides.

Study of Förster Resonance Energy Transfer to Lipid Domain Markers Ascertains Partitioning of Semisynthetic Lipidated N-Ras in Lipid Raft Nanodomains.

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (also known as "lipid rafts") surrounded by the liquid-disordered phase. Many membrane-associated proteins were found to permanently integrate into the lipid rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been demonstrated in vitro in model bilayers with recombinant proteins and therefore has been attributed to the action of binding of Ras to other proteins at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in lipid raft vesicles in the absence of additional proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster resonance energy transfer between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized into the liquid-disordered domains. Herein, we established that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing its preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found to be localized outside of the liquid-ordered rafts, most likely in the bulk-disordered lipid. Substitution of the N-terminal G domain of N-Ras with a homologous G domain of H-Ras disrupted the nucleotide-dependent lipid domain switch.