Characterization of Arthropeptide B, an Antifungal Cyclic Tetrapeptide from Arthrobacter humicola.

Disease suppressive composts are known, yet little information on the potential role of specific microbial antagonist within are available. Arthrobacter humicola isolate M9-1A has been obtained from a compost prepared from marine residues and peat moss. The bacterium is a non-filamentous actinomycete with antagonistic activity against plant pathogenic fungi and oomycetes sharing its ecological niche in agri-food microecosystems. Our objective was to identify and characterize compounds with antifungal activity produced by A. humicola M9-1A. Arthrobacter humicola culture filtrates were tested for antifungal activity in vitro and in vivo and a bioassay-guided approach was used to identify potential chemical determinants of its observed activity against molds. The filtrates reduced the development of lesions of Alternaria rot on tomatoes and the ethyl acetate extract inhibited growth of Alternaria alternata. A compound, arthropeptide B [cyclo-(L-Leu, L-Phe, L-Ala, L-Tyr)], was purified from the ethyl acetate extract of the bacterium. Arthropeptide B is a new chemical structure reported for the first time and has shown antifungal activity against A. alternata spore germination and mycelial growth.

Biological and chemical characterization of antimicrobial activity in Arthrobacter spp. isolated from disease-suppressive compost.

Antagonistic bacteria can act as biocontrol agents against various phytopathogens. Recently, Arthrobacter spp. demonstrated antifungal activity, but were not further characterized. In this study, the antimicrobial activity of Arthrobacter humicola strains M9-1A, M9-2, and M9-8, and Arthrobacter psychrophenolicus strain M9-17 were evaluated against nine plant pathogens in vitro, and their cell-free filtrates were additionally assessed for inhibition of Alternaria alternata and suppression of black mold disease on tomato fruit. Results indicated that A. humicola M9-1A and A. psychrophenolicus M9-17 were the most inhibitory, reducing growth of seven of the pathogens studied. Cell-free filtrates of A. psychrophenolicus M9-17 reduced the growth of most pathogens. All cell-free bacterial filtrates, except those from A. humicola M9-2, suppressed black mold on tomato fruit. Disk diffusion assays with ethyl acetate soluble culture filtrate extracts of all bacteria reduced the mycelial growth of A. alternata. Clear inhibition zones were observed for A. psychrophenolicus M9-17 extracts using drop bioassays. The antifungal compound N-acetyltryptamine was purified and characterized from the A. psychrophenolicus M9-17 cell-free ethyl acetate soluble extract. This study suggests that antibiosis may play a key role in the antimicrobial activity of Arthrobacter spp.

Role of Lipid Composition in the Interaction and Activity of the Antimicrobial Compound Fengycin with Complex Membrane Models.

Fengycins are compounds produced by bacteria of the Bacillus genus with strong antifungal activity. In this work, lipids extracted from fungal and oomycetal molds were used to assess the ability of fengycin to bind and insert into complex membrane models prepared as Langmuir lipid monolayers. In addition, fengycin-induced leakage in liposomes prepared from these complex lipid extracts was also evaluated. Fengycin's ability to bind and incorporate into these membranes seemed to be mainly related to ergosterol content. Other membrane characteristics such as phospholipid fatty acyl chain length played a more peripheral role. A high ergosterol concentration appeared to allow other membrane characteristics generally associated with fengycin binding and/or insertion, such as higher proportion of phosphatidylcholine head groups or increased fatty acyl unsaturation, to be present without adversely affecting membrane integrity. Increased membrane leakage was also generally associated with the presence of low or no ergosterol. Leakage was also correlated with the previously reported biological activity of fengycin on these molds.

Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions.

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9-5.

Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9-5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9-5 were quantified over the entire period of microbial interaction. B. subtilis B9-5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens' overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9-5 protecting its ecological niche against the ingress of these pathogens.

Physiological and molecular characterization of compost bacteria antagonistic to soil-borne plant pathogens.

Disease suppressive composts have the potential to mitigate the risks associated with chemical pesticides. One of the main characteristics responsible for the suppressive nature of composts is their microbiological populations. To gain insight into the determinants responsible for their suppressive effects, we assayed composts to (i) isolate and identify beneficial antagonistic bacteria, (ii) quantify their antifungal and anti-oomycetal activities, (iii) extract inhibitory compounds produced by the bacteria, and (iv) identify antimicrobial lipopeptides produced by these bacteria. The antagonistic bacteria belonged to the genera Arthrobacter, Pseudomonas, Bacillus, Brevibacillus, Paenibacillus, and Rummeliibacillus and had the ability to antagonise the growth of Fusarium sambucinum, Verticillium dahliae, and (or) Pythium sulcatum. These bacteria produced antimicrobial compounds that affected the mycelial growth and (or) conidial germination of the pathogens. Mass spectrometry analyses showed the presence of various antimicrobial lipopeptides in Bacillus and Bacillus-related spp. extracts, demonstrating that they are responsible, at least in part, for the antagonistic activity of the bacteria. Results from this work provide greater insight into some of the biological, biochemical, and physiological determinants of suppressiveness in composts involved in the control of plant pathogens.

Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization.

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

Effect of tea tree (Melaleuca alternifolia) oil as a natural antimicrobial agent in lipophilic formulations.

There has been increased interest surrounding the use of tea tree oil (TTO) as a natural antimicrobial. In this study, the antimicrobial activity of TTO and its components were investigated in vitro and in a predominantly lipid-based personal care formulation. In vitro, TTO showed minimal inhibitory concentrations of 0.2% (for Saccharomyces cerevisiae and Pythium sulcatum), 0.4% (for Escherichia coli, Bacillus subtilis, and Rhizopus stolonifer), and 0.8% (for Botrytis cinerea). TTO at 0.08%-0.8% was often as efficient as parabens. Comparison of the antimicrobial activities of TTO components showed that terpinen-4-ol and γ-terpinene were generally most effective in inhibiting microbial growth. TTO activity in a personal care product was evaluated through air and water exposure, artificial inoculation, and shelf life studies. While TTO did not increase shelf life of unopened products, it decreased microbial load in products exposed to water and air. Results from this study support that antimicrobial activity of TTO can be attributed to varying levels of its components and that low levels of TTO were effective in reducing microbial growth during the use of the product. This study showed that TTO can act as a suitable preservative system within an oil-based formulation.

Regulation of the Rana sylvatica brevinin-1SY antimicrobial peptide during development and in dorsal and ventral skin in response to freezing, anoxia and dehydration.

Brevinin-1SY is the only described antimicrobial peptide (AMP) of Rana sylvatica. As AMPs are important innate immune molecules that inhibit microbes, this study examined brevinin-1SY regulation during development and in adult frogs in response to environmental stress. The brevinin-1SY nucleotide sequence was identified and used for protein modeling. Brevinin-1SY was predicted to be an amphipathic, hydrophobic, alpha helical peptide that inserts into a lipid bilayer. Brevinin-1SY transcripts were detected in tadpoles and were significantly increased during the later stages of development. Effects of environmental stress (24 h anoxia, 40% dehydration or 24 h frozen) on the mRNA levels of brevinin-1SY in the dorsal and ventral skin were examined. The brevinin-1SY mRNA levels were increased in dorsal and ventral skin of dehydrated frogs, and in ventral skin of anoxic frogs, compared with controls (non-stressed). Brevinin-1SY protein levels in peptide extracts of dorsal skin showed a similar, but not significant, trend to that of brevinin-1SY mRNA levels. Antimicrobial activity of skin extracts from control and stressed animals were assessed for Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Botrytis cinerea, Rhizopus stolonifer and Pythium sulcatum using disk diffusion assays. Peptide extracts of dorsal skin from anoxic, frozen and dehydrated animals showed significantly higher inhibition of E. coli and P. sulcatum than from control animals. In ventral skin peptide extracts, significant growth inhibition was observed in frozen animals for E. coli and P. sulcatum, and in anoxic animals for B. cinerea, compared with controls. Environmental regulation of brevinin-1SY may have important implications for defense against pathogens.

Cellular Lipid Composition Affects Sensitivity of Plant Pathogens to Fengycin, an Antifungal Compound Produced by Bacillus subtilis Strain CU12.

Fengycin is an antimicrobial cyclic lipopeptide produced by various Bacillus subtilis strains, including strain CU12. Direct effects of fengycin include membrane pore formation and efflux of cellular contents leading to cell death in sensitive microorganisms. In this study, four plant pathogens were studied in order to elucidate the role of membrane lipids in their relative sensitivity to fengycin. Inhibition of mycelial growth in these pathogens varied considerably. Analysis of membrane lipids in these microorganisms indicated that sensitivity correlated with low ergosterol content and shorter phospholipid fatty acyl chains. Sensitivity to fengycin also correlated with a lower anionic/zwitterionic phospholipid ratio. Our data suggest that decreased fluidity buffering capacity, as a result of low ergosterol content, and higher intrinsic fluidity afforded by short fatty acyl chain length may increase the sensitivity of microbial membranes to fengycin. Our results also suggest that lower content in anionic phospholipids may increase fengycin insertion into the membrane through reduced electrostatic repulsion with the negatively charged fengycin. The intrinsic membrane lipid composition may contribute, in part, to the observed level of antimicrobial activity of fengycin in various plant pathogens.

Investigating the effects of L- to D-amino acid substitution and deamidation on the activity and membrane interactions of antimicrobial peptide anoplin.

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, anoplin is the smallest linear α-helical antimicrobial peptide found naturally with broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes. Deamidation was found to decrease the peptide's antibacterial properties. In the present work, interactions of amidated (Ano-NH2) and deamidated (Ano-OH) forms of anoplin as well as Ano-NH2 composed of all D-amino acids (D-Ano-NH2) with model cell membranes were investigated by means of Langmuir Blodgett (LB) technique, atomic force microscopy (AFM), X-ray photoemission electron microscopy (X-PEEM) and carboxyfluorescein leakage assay in order to gain a better understanding of the effect of these peptide modifications on membrane binding and lytic properties. According to LB, all three peptides form stable monolayers at the air/water interface with Ano-NH2 occupying a slightly greater area per molecule than Ano-OH. All three forms of the peptide interact preferentially with anionic 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), rather than zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. Peptides form nanoscale clusters in zwitterionic but not in anionic monolayers. Finally, membrane lytic activity of all derivatives was found to depend strongly on membrane composition and lipid/peptide ratio. The results suggest that amidated forms of peptides are likely to possess higher membrane binding affinity due to the increased charge.

Arthropeptide A, an antifungal cyclic tetrapeptide from Arthrobacter psychrophenolicus isolated from disease suppressive compost.

In an effort to describe bioactive antifungal compounds from antagonistic bacteria with potential for biocontrol of plant pathogens, a strain of Arthrobacter psychrophenolicus was collected from plant disease suppressive compost prepared from composted material of marine origin. Few natural products have been characterized from the non-filamentous Actinobacteria genus Arthrobacter. A new cyclic tetrapeptide, cyclo-(L-Pro-L-Leu-L-γHyp-L-Tyr); arthropeptide A (1), was isolated from the EtOAc soluble culture filtrate extract of A. psychrophenolicus M9-17 grown in MOLP broth. Its structure was confirmed by HRMS, interpretation of NMR data, and a modified Marfey's method. Arthropeptide A (1) displayed antifungal activity towards Alternaria alternata, the causal agent of disease in numerous host plant species, which had shown the previous susceptibility to A. psychrophenolicus. The newly identified compound may be responsible, in part, for the inhibitory activity of the bacterium against fungal plant pathogens.

Supported lipid bilayers using extracted microbial lipids: domain redistribution in the presence of fengycin.

Fengycin is an antimicrobial cyclic lipopeptide known to interact with microbial cell membranes. To gain insight into the role of lipids in fengycin sensitivity, lipids extracted from Alternaria solani, Fusarium sambucinum, and Pythium sulcatum were analyzed and used in the preparation of supported lipid bilayers (SLBs). Total Internal Reflection Fluorescence Microscopy (TIRFM) was used to evaluate lipid phase separation within the SLBs and changes in domain distribution with the application of fengycin. A. solani lipid extract contained the highest quantity of ergosterol while P. sulcatum contained no ergosterol. Sterol content of SLBs was strongly correlated with increases in phase separation, suggesting ergosterol may play a role in promoting ordering of the lipid phases in the bilayer. A. solani experienced the least change in domain characteristics following fengycin exposure, suggesting ergosterol may be buffering effects of the antimicrobial compound. Factors such as lipid headgroup charge and unsaturation levels may impact fengycin's effects on domain phase separation, but these effects were generally overshadowed by the role of ergosterol. In the absence of ergosterol, in the P. sulcatum bilayers, fengycin caused an increase in lipid phase ordering.

Domain redistribution within ergosterol-containing model membranes in the presence of the antimicrobial compound fengycin.

The cyclic lipopeptide fengycin, produced by Bacillus subtilis, exhibits its antimicrobial capabilities by altering the integrity of the cell membrane of plant pathogens. Previous work has correlated fengycin activity with membrane characteristics, such as sterol content. This work focused on the influence of fengycin on supported lipid bilayers containing varying levels of ergosterol. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and distinguish ordered (Lß/Lo) and disordered (Lα/Ld) domains in the model membranes following exposure to low (50 µg) and high (500 µg) fengycin doses. Application of an initial low dose of fengycin to 0% and 3% ergosterol-containing bilayers resulted in redistribution of Lα/Lß and Lo/Ld domains, respectively, which the bilayers compensated and corrected for over time. These membranes were unable to tolerate a second 50 µg dose or a single high fengycin dose. The 6% ergosterol bilayers were able to tolerate sequential low doses of fengycin. Exposure of these bilayers to the high fengycin dose caused a decrease in the number of Lo domains, albeit less than that seen in the 0% and 3% ergosterol bilayers. Bilayers containing 12% ergosterol, exhibited the least amount of change after fengycin exposure. These were the only bilayer to exhibit an increase in area taken up by ordered domains. These results suggest fengycin may preferentially act on the Lß or Lo phase, the area in which ergosterol resides. Bilayers containing low levels of ergosterol appear to be more sensitive to the lipopeptide, suggesting ergosterol plays a role in buffering perturbations caused by fengycin.

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system.

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.

Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide.

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

Microbial inhibitors of the fungus Pseudogymnoascus destructans, the causal agent of white-nose syndrome in bats.

Pseudogymnoascus destructans, the fungus that causes white-nose syndrome in hibernating bats, has spread across eastern North America over the past decade and decimated bat populations. The saprotrophic growth of P. destructans may help to perpetuate the white-nose syndrome epidemic, and recent model predictions suggest that sufficiently reducing the environmental growth of P. destructans could help mitigate or prevent white-nose syndrome-associated bat colony collapse. In this study, we screened 301 microbes from diverse environmental samples for their ability to inhibit the growth of P. destructans. We identified 145 antagonistic isolates, 53 of which completely or nearly completely inhibited the growth of P. destructans in co-culture. Further analysis of our best antagonists indicated that these microbes have different modes of action and may have some specificity in inhibiting P. destructans. The results suggest that naturally-occurring microbes and/or their metabolites may be considered further as candidates to ameliorate bat colony collapse due to P. destructans.

Combating Antimicrobial Resistance in Foodborne Microorganisms.

This review addresses an important public health hazard affecting food safety. Antimicrobial agents are used in foods to reduce or eliminate microorganisms that cause disease. Many traditional organic compounds, novel synthetic organic agents, natural products, peptides, and proteins have been extensively studied for their effectiveness as antimicrobial agents against foodborne Campylobacter spp., Escherichia coli, Listeria spp. and Salmonella. However, antimicrobial resistance can develop in microorganisms, enhancing their ability to withstand the inhibiting or killing action of antimicrobial agents. Knowledge gaps still exist with regard to the actual chemical and microbiological mechanisms that must be identified to facilitate the search for new antimicrobial agents. Technical implementation of antimicrobial active packing films and coatings against target microorganisms must also be improved for extended product shelf life. Recent advances in antimicrobial susceptibility testing can provide researchers with new momentum to pursue their quest for a resistance panacea.

Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens.

Bacillus subtilis cyclic lipopeptides are known to have various antimicrobial effects including different types of interactions with the cell membranes of plant pathogenic fungi. The various spectra of activities of the three main lipopeptide families (fengycins, iturins, and surfactins) seem to be linked to their respective mechanisms of action on the fungal biomembrane. Few studies have shown the combined effect of more than one family of lipopeptides on fungal plant pathogens. In an effort to understand the effect of producing multiple lipopeptide families, sensitivity and membrane permeability of spores from four fungal plant pathogens (Alternaria solani, Fusarium sambucinum, Rhizopus stolonifer, and Verticillium dahliae) were assayed in response to lipopeptides, both individually and as combined treatments. Results showed that inhibition of spores was highly variable depending on the tested fungus-lipopeptide treatment. Results also showed that inhibition of the spores was closely associated with SYTOX stain absorption suggesting effects of efficient treatments on membrane permeability. Combined lipopeptide treatments revealed additive, synergistic or sometimes mutual inhibition of beneficial effects.