Exopolysaccharides of Paenibacillus polymyxa: A review.

Paenibacillus polymyxa (P. polymyxa) is a member of the genus Paenibacillus, which is a rod-shaped, spore-forming gram-positive bacterium. P. polymyxa is a source of many metabolically active substances, including polypeptides, volatile organic compounds, phytohormone, hydrolytic enzymes, exopolysaccharide (EPS), etc. Due to the wide range of compounds that it produces, P. polymyxa has been extensively studied as a plant growth promoting bacterium which provides a direct benefit to plants through the improvement of N fixation from the atmosphere and enhancement of the solubilization of phosphorus and the uptake of iron in the soil, and phytohormones production. Among the metabolites from P. polymyxa, EPS exhibits many activities, for example, antioxidant, immunomodulating, anti-tumor and many others. EPS has various applications in food, agriculture, environmental protection. Particularly, in the field of sustainable agriculture, P. polymyxa EPS can be served as a biofilm to colonize microbes, and also can act as a nutrient sink on the roots of plants in the rhizosphere. Therefore, this paper would provide a comprehensive review of the advancements of diverse aspects of EPS from P. polymyxa, including the production, extraction, structure, biosynthesis, bioactivity and applications, etc. It would provide a direction for future research on P. polymyxa EPS.

Dietary Paenibacillus polymyxa AM20 as a new probiotic: Improving effects on IR broiler growth performance, hepatosomatic index, thyroid hormones, lipid profile, immune response, antioxidant parameters, and caecal microorganisms.

The search for a natural antimicrobial agent is ongoing and critical because of the rise and rapid proliferation of antibiotic-resistant pathogenic bacteria. The current study aims to examine the effect of Paenibacillus polymyxa AM20 as an alternative antibiotic and feed additive on Indian river broiler performance, digestive enzymes, thyroid hormones, lipid profile, hepatosomatic index, immunological response, gut bacteria, and antioxidant parameters. The bacterial isolate AM20 was identified at the gene level by isolating DNA and using PCR to detect genes. Based on 16S rRNA gene sequence analysis, the bacterial isolate was identified as Paenibacillus polymyxa. One hundred twenty Indian river broilers (1-day old) were randomly divided into 4 groups of 10 chicks each, with 3 replicates. The control group was fed a basal diet only, while the other 3 were administered control diets supplemented with P. polymyxa at 3 concentrations: 0.5, 1, and 1.5 mg/kg. The findings revealed that all groups that received graded amounts of P. polymyxa increased all growth parameters throughout the study. P. polymyxa treatment at 1.5 mg/kg increased body gain by 9% compared to the control due to increased feed intake (P = 0.0001), growth rate (P = 0.0001), and decreased feed conversion ratio. Compared to the control group, P. polymyxa (1.5 mg/kg) enhanced kidney functions in chickens by reducing uric acid and creatinine levels (P = 0.0451). Compared to the control group, alanine aminotransferase and aspartate transaminase levels in the liver were significantly reduced at all P. polymyxa doses. Liver function values were highest for P. polymyxa at 1.5 mg/kg. Compared to the control group, those whose diets included P. polymyxa had significantly better blood cholesterol levels, high-density lipoprotein, low-density lipoprotein, immunological response, thyroid function, and gut microbiota. In general, broiler chickens' economic efficiency was improved by including P. polymyxa in their diet, which also improved their growth performance, carcass dressing, specific blood biochemical levels and enzymes, and the composition of the gut microbiota.

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

Biological activity of silver nanoparticles synthesized with Paenibacillus polymyxa exopolysaccharides.

Recently, there has been increased interest in the synthesis of nanoparticles by using natural polysaccharides. These polysaccharides are eco-friendly, nontoxic, and cheap to prepare. On the other hand, the attention in hydrocolloids and films has significantly enhanced, and their application is very promising in the food, pharmaceutical, perfumery and cosmetics, oil, paper, and textile industries. In this context, the present study is aimed to prepare silver nanoparticles by using viscous and superviscous exopolysaccharides of the rhizobacterium Paenibacillus polymyxa strains, CCM 1465 and 88A, and examined the properties of the resultant nanoparticles. We examined the synthesis and properties of silver nanoparticles under variable synthetic conditions by using exopolysaccharides of the rhizobacteria Paenibacillus polymyxa CCM 1465 and 88A. To prepare nanoparticles, we used different combinations of exopolysaccharide and silver nitrate concentrations: 1-10 mg/mL and 1-40 mM, respectively. The resulting solutions were alkalinized from pH 7.5-12 and heated for 15, 30, and 60 min to determine the optimal synthetic conditions. We found that the exopolysaccharides of strains CCM 1465 and 88A reduced silver ions and acted as nanoparticle stabilizers. The prepared spherical, oval, and triangular particles were stable and ranged in size from 2 to 40 nm, depending on the strain and on the experimental conditions. The nanoparticles showed antibacterial and antifungal activity against Escherichia coli K-12, Pseudomonas aeruginosa 50.3, Bacillus subtilis 26-D, and Fusarium oxysporum. In addition, the nanoparticles were active against SK-MEL-2 human melanoma cells. This finding shows the promise of further research on the exopolysaccharides of P. polymyxa 1465 and 88А in different fields of science, including medicine.

Investigation of bioactivity of unsaturated oligo­galacturonic acids produced from apple waste by Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 Pectinases.

Pectin is one of the main structural components in fruits and an indigestible fiber made of D-galacturonic acid units with α (1-4) linkage. This study investigates the microbial degradation of pectin in apple waste and the production of bioactive compounds. Firstly, pectin-degrading bacteria were isolated and identified, then pectinolytic activity was assessed by DNS. The products were evaluated by TLC and LC-MS-ESI. The antioxidative effects were investigated using DPPH and anti-cancer effects and cytotoxicity were analyzed by MTT and flow cytometry. In this study two new bacterial isolates, Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 with the pectinolytic enzyme were introduced. Structure analysis showed that the products of enzymatic degradation include unsaturated mono, di, tri, and penta galacturonic acids with 74% and 69% RSA at 40 mg/mL for A. faecalis and P. polymyxa S4, respectively. The results of anti-tumor properties on MCF-7 cells by MTT assay, for products of AGS3 and S4 at 40 mg/mL after 48 h, showed 7% and 9% survival, respectively. In the flow cytometric assessment, the compounds of AGS3 at 40 mg/mL were 100% lethal in 48 h and regarding S4 isolate caused 98% death. Cytotoxicity evaluation on L-929 cells showed no significant toxicity on living cells.

Antigenotoxic potential of the fermentation broth produced by Paenibacillus polymyxa RNC-D in vitro.

Aim: Evaluate the chemopreventive potential of the extract from P. polymyxa RNC-D. Methods: Concentrations of P. polymyxa RNC-D extract were tested in HepG2/C3A cells to assess their genotoxic (comet assay), mutagenic (micronucleus test) and antigenotoxic potential (comet assay) in vitro. Results: 400 and 40 µg/ml concentrations induced DNA lesions, whereas the 4 µg/ml induced a desmutagenic effect. Complementary tests indicated that the extract minimized the formation of reactive oxygen species induced by methyl methanesulfonate and normalized the loss of membrane potential. The quantification of cytokines indicated that TNF-α was immunostimulated by the extract. However, when administered in conjunction with the methyl methanesulfonate, the extract blocked the TNF-α release. Conclusion: The fermentation broth from P. polymyxa RNC-D showed an antigenotoxic effect, and thus the potential to be used as chemopreventive compound.

Isolation, characterization and cytoprotective effects against UV radiation of exopolysaccharide produced from Paenibacillus polymyxa PYQ1.

A novel exopolysaccharide (EPS) from Paenibacillus polymyxa PYQ1 was extracted, well purified and characterized. This EPS was homogeneous glucomannan-type polysaccharide with the average molecular weight of 4.38 × 106 Da. Structural characterization indicated that the monosaccharides of EPS were pyranoses connected by ß-glycosidic linkages. Furthermore, our results showed the protective benefits of EPS against UVC induced cytotoxicity in HaCaT cells through scavenging excessive reactive oxygen species, mitigating the decrease of mitochondrial membrane potential, improving catalase activity and maintaining membrane integrity. Taken together, this study qualified EPS from P. polymyxa PYQ1 was a promising natural polymer which worth further investigation as a skin-care agent.

Transfer of Nitrogen Fixation (nif) Genes to Non-diazotrophic Hosts.

Nitrogen is one of the most important nutrients for plant growth. To enhance crop productivity, chemical nitrogen fertilizer is commonly applied in agriculture. Biological nitrogen fixation, the conversion of atmospheric N2 to NH3 , is an important source of nitrogen input in agriculture and represents a promising substitute for chemical nitrogen fertilizers. However, nitrogen fixation is only sporadically distributed within bacteria and archaea (diazotrophs). Thus, many biologists hope to reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host, with the final aim of developing N2 -fixing cereal crops. With the advent of synthetic biology and a deep understanding of the fundamental genetic determinants necessary to sustain nitrogen fixation in bacteria, much progress has been made toward this goal. Transfer of native and refactored nif (nitrogen fixation) genes to non-diazotrophs has been attempted in model bacteria, yeast, and plants. Specifically, nif genes from Klebsiella oxytoca, Azotobacter vinelandii, and Paenibacillus polymyxa have been successfully transferred and expressed in Escherichia coli, Saccharomyces cerevisiae, and even in the tobacco plant. These advances have laid the groundwork to enable cereal crops to "fix" nitrogen themselves to sustain their growth and yield.

In silico identification and characterization of antineoplastic asparaginase enzyme from endophytic bacteria.

It is generally accepted that L-asparagine is an important amino acid required for the fast growth of cells. Cancerous cells receive this amino acid from extracellular sources. The depletion of L-asparagine from its surrounding environments by asparaginase enzyme can be used as a therapeutic strategy in cancer patients. This therapeutic enzyme is produced commercially mainly from bacteria such as Escherichia coli and Erwinia chrysanthemi. The side effects of such drugs have persuaded scientists to find new enzyme sources. In this study, in silico approach was applied to investigate L-asparaginase producing endophytic bacteria that produce more compatible enzymes within the body. Protein-protein basic local alignment search tool with E. coli and E. chrysanthemi asparaginase enzyme sequences against 262 endophytic bacteria were performed. The results with identity more than 35%, coverage more than 80%, and E-value less than 10-4 were selected. Then, some of bioinformatics tools were used to characterize them. A total of nine sequences consisting of seven known and two hypothetical proteins were identified in six bacterial species. The results showed that some of the asparaginase enzymes produced by endophytic bacteria possess more suitable immunological indices compared with asparaginase enzymes of E. coli and E. chrysanthemi. Herbaspirillum rubrisubalbicans was predicted to produce a nonallergen and nonantigen asparaginase enzyme. The number of antigenic determinants was predicted to be lower in asparaginase enzymes produced by Bacillus amyloliquefaciens, H. rubrisubalbicans, and H. seropedicae. Moreover, the number of high-scored B-cell epitopes was lower in enzyme sequences related to the mentioned bacteria and Paenibacillus polymyxa. The number of discontinuous epitopes and the number of T-cell epitopes were lower in B. amyloliquefaciens produced enzymes. Therefore, the therapeutic use of these enzymes is possible.

Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: A proposal for biocontrol of phytopathogenic fungi.

Biocontrol has emerged in recent years as an alternative to pesticides. Given the importance of environmental preservation using biocontrol, in this study two antagonistic bacteria against phytopathogenic fungi were isolated and evaluated. These bacterial strains, identified as Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24, inhibited (70 to 80%) the development of two phytopathogens of economic importance: the fungus Rhizoctonia solani RhCh-14, isolated from chili pepper, and the oomycete Pythium ultimum PyFr-14, isolated from tomato. The spectrum was not limited to the previous pathogens, but also to other phytopathogenic fungus, some bacteria and other oomycetes. Fungi-bacteria microcultures observed with optical and scanning electron microscopy revealed hyphae disintegration and pores formation. The antifungal activity was found also in the supernatant, suggesting a diffusible compound is present. Innocuous tests on tobacco leaves, blood agar, bean seed germination and in Galleria mellonella larvae showed that strain NMA1017 has the potential to be a biocontrol agent. Greenhouse experiments with bean plants inoculated with P. polymyxa exhibited the efficacy to inhibit the growth of R. solani and P. ultimum. Furthermore, P. polymyxa NMA1017 showed plant growth promotion activities, such as siderophore synthesis and nitrogen fixation which can contribute to the crop development.

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells.

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b-LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies.

Reconstitution and Substrate Specificity of the Thioether-Forming Radical S-Adenosylmethionine Enzyme in Freyrasin Biosynthesis.

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the ß- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the ß-carbon of Asp (S-Cß). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

Study on the simultaneous degradation of five pesticides by Paenibacillus polymyxa from Panax ginseng and the characteristics of their products.

The quality and safety of ginseng products were seriously affected due to the slow metabolism and long-term residual pesticides in ginseng. Microbial degradation is an effective method to degrade pesticide residues. In this study, ginseng endophytic Paenibacillus polymyxa was used to degrade pesticide residues. A method of simultaneous determination of fluazinam, BHC, PCNB, chlorpyrifos and DDT in ginseng roots and ginseng stems and leaves by GC was established. The sample was extracted with n-hexane and purified by Florisil solid phase extraction column. The limit of quantitation was 0.01 µg mL-1, the linear relationship was good (r ≥ 0.9901). 7 days after inoculated with P. polymyxa, the degradation rates of fluazinam, BHC, PCNB, chlorpyrifos, and DDT in the medium were 94.77%, 70.34%, 77.92%, 78.30%, 66.70%, respectively (P < 0.05). The safety of 5 pesticide degradation products was investigated by GC-MS. The results showed that after 7 days degradation, the main degradation products were alkanes, which are non-toxic and can't cause secondary pollution to the environment. The actual degradation results were verified by field experiments. The results indicated that after sprayed 5 times with P. polymyxa, the degradation rates of fluazinam, BHC, PCNB, chlorpyrifos and DDT in the ginseng roots were 66.07%, 46.24%, 21.05%, 72.40%, 54.21%, respectively (P < 0.05). The degradation rates in ginseng stems and leaves were 74.18%, 55.61%, 73.65%, 58.13%, 46.91%, respectively (P < 0.05). The results indicated that Paenibacillus polymyxa was an effective degradation strain of 5 pesticides.

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

An endophytic strain of genus Paenibacillus isolated from the fruits of Noni (Morinda citrifolia L.) has antagonistic activity against a Noni's pathogenic strain of genus Aspergillus.

Endophytes are microbes capable of colonizing the tissues of healthy plants and subsequently establishing a harmonious relationship with their hosts. In this research, the endophytic strain Paenibacillus sp. NEB was isolated from fruits of healthy Noni (Morinda citrifolia L.). Strain NEB was identified as Paenibacillus polymyxa using MALDI-TOF Mass Spectrometry. Pathogenic fungal strain NP-1 was isolated from Noni fruits infected by smut, and was identified as Aspergillus aculeatus by polyphasic taxonomy basing on morphological identification, and ITS-5.8S rDNA and ß-tubulin gene phylogenetic analyses. Through the antagonistic test against the pathogenic strain Aspergillus aculeatus NP-1, the results showed that strain NEB had a good antagonistic activity against smut pathogen of Noni. By sequencing with Illumina HiSeq 2000, the draft genome of Paenibacillus sp. NEB was acquired, and 3 CDSs for glucanases were annotated and potentially correlated to the antagonistic activity of this strain. Using realtime-PCR method with specific primers to amplify the biocontrol gene, ß-1,3-1,4- glucanase gene (gluB), it was found in Paenibacillus polymyxa NEB. This study would provide a theoretical and microbial basis for the rationally developing and using Noni beneficial microbial inoculants against its pathogenic strain in the future.

Immunomodulatory activity of exopolysaccharide from the rhizobacterium Paenibacillus polymyxa CCM 1465.

Bacterial polysaccharides are promising stimulants of protective functions in humans and animals. We investigated the ability of exopolysaccharide from the rhizobacterium Paenibacillus polymyxa CCM 1465 to induce nonspecific resistance factors in the macroorganism. We examined in vitro the effect of the exopolysaccharide, produced with different carbon sources, on the phagocytic activity of murine macrophages, on the generation of reactive oxygen species and of enzymes (acid phosphatase and myeloperoxidase), on the proliferation of murine splenocytes, and on the synthesis of proinflammatory cytokines [interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)] by human mononuclear cells. The exopolysaccharide promoted the phagocytosis of bacterial cells, activated metabolic processes in human and animal leukocytes, and moderately affected the production of TNF-α and IL-1ß. The exopolysaccharides produced on media with glucose and sucrose differed in their effect on the immune cells, possibly owing to their different compositions, structures, and properties. The results validly indicate that the exopolysaccharide of P. polymyxa CCM 1465 promotes nonspecific immunity. Therefore, it can find application as a biologically active immunomodulatory substance.

Cytotoxic Effects and Production of Cytokines Induced by the Endophytic Paenibacillus polymyxa RNC-D In Vitro.

BACKGROUND: Prominent among all the organisms that have a potential value for the production of new medicines, are endophytes, fungi and bacteria that live inside plants without harming them. In this study, a total lyophilized extract (TLE) of Paenibacillus polymyxa RNC-D was used. The P. polymyxa lineages are known for their capacity to segregate a large number of extracellular enzymes and bioactive substances. METHODS: The TLE of Paenibacillus polymyxa RNC-D was tested in cell viability assays for cytotoxicity and cytokine production in BALB/3T3 and J774A.1 cell lineages. RESULTS: A 50% mortality rate of fibroblasts (BALB/3T3) was observed in the 1.171±0.161 mg/mL and 0.956±0.112 mg/mL doses after 48 and 72 hours, respectively, as well as a 50% mortality rate of macrophage cells (J774A.1) in the 0.994±0.170 mg/mL and 0.945±0.280 mg/mL doses after 48 and 72 hours, respectively. The ≈1 mg/mL concentration significantly affected the kinetic of growth in all the measured periods. The extract induced apoptosis and necrosis 24 hours after the ≈1 mg/mL concentration in both tested lineages. The treatment with the ≈1 mg/mL concentration led to the production of TNF-α and IFN-γ cytokines in 24 hours. IL-12 and IL-10 began to be detected as a result of the treatment with 0.1 mg/mL. However, with the 0.5 mg/mL dose in 24 hours, a significant reduction in IL-10 was observed. CONCLUSION: Our data suggest that the TLE of P. polymyxa RNC-D modulated the production of cytokines with different patterns of immune response in a dose-dependent way.

Mechanism of action of AMP-jsa9, a LI-F-type antimicrobial peptide produced by Paenibacillus polymyxa JSa-9, against Fusarium moniliforme.

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32µg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K+, protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries.

The toxic mode of action of cyclic lipodepsipeptide fusaricidins, produced by Paenibacillus polymyxa, toward mammalian cells.

AIMS: Toxigenic strains of Paenibacillus polymyxa were isolated from buildings connected with the symptoms of ill health. Our aim was to identify the toxic compounds of Paenibacillus polymyxa and to describe their toxic actions. METHODS AND RESULTS: The toxins of Paenibacillus polymyxa were purified and analysed by HPLC and mass spectrometry. Toxic fusaricidins A and B, and LI-F05a with mass ions at m/z 883·7, 897·6 and 897·6, respectively, were found. The cytotoxicity of purified fusaricidins A and B was measured using boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts. The ion channel forming properties of fusaricidins were studied using the black lipid membrane (BLM) technique. Fusaricidins A and B depolarized the mitochondria of boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts at concentrations of 0·5-1 µg ml-1 and caused nuclear fragmentation and induced apoptosis at concentrations of 2·5-5 µg ml-1 . Furthermore, fusaricidins A and B induced K+ permeating single channels. CONCLUSIONS: It was concluded that fusaricidins were toxic to mitochondria and induced apoptosis in mammalian cells. It was proposed that the observed toxicity of fusaricidins is due their ion channel forming properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper revealed, for the first time, the mode of action of Paenibacillus polymyxa fusaricidins toxins towards mammalian cells. Fusaricidins, due to their potassium ionophoricity and mitochondria depolarizing impacts, may have contributed to the health damage observed at sites where the producer strains were isolated at high density.