Application of extracellular polymers on soil communities exposed to oil and nickel contamination.

The petrochemical industry is responsible for many accidental releases of pollutants in soil such as hydrocarbons and toxic metals. This co-contamination is responsible for a delay in the degradation of the organic pollution. Many successful technologies to remove these metals apply extracellular polymeric substances (EPS). In this study, we tested the application of an EPS from a Paenibacillus sp. to aid the bioremediation of soils contaminated with crude oil and nickel. We conducted a microcosm experiment to soils containing combinations of oil, nickel, and EPS. The final concentration of oil was evaluated with an infrared spectrometer. Also, we sequenced the metagenomes of the samples in an ion torrent sequencer. The application of EPS did not aid the removal of hydrocarbons with or without the presence of nickel. However, it led to a smaller decrease in the diversity indexes. EPS decreased the abundance of Actinobacteria and increased that of Proteobacteria. The EPS also decreased the connectivity among Actinobacteria in the network analysis. The results indicated that the addition of EPS had a higher effect on the community structure than nickel. Altogether, our results indicate that this approach did not aid the bioremediation of hydrocarbons likely due to its effect in the community structure that affected hydrocarbonoclastic microorganisms.

Paenibacillus puerhi sp. nov., isolated from the rhizosphere soil of Pu-erh tea plants (Camellia sinensis var. assamica).

An aerobic, Gram-staining-positive, rod-shaped, endospore-forming and motile bacterial strain, designated SJY2T, was isolated from the rhizosphere soil of tea plants (Camellia sinensis var. assamica) collected in the organic tea garden of the Jingmai Pu-erh tea district in Pu'er city, Yunnan, southwest China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Paenibacillus. The closest phylogenetic relative was Paenibacillus filicis DSM 23916T (98.1% similarity). The major fatty acids (> 10% of the total fatty acids) were anteiso-C15:0 and isoC16:0. The major respiratory quinone was MK-7 and the major polar lipid was diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The peptidoglycan contained glutamic acid, serine, alanine and meso-diaminopimelic acid. Genome sequencing revealed a genome size of 6.71 Mbp and a G + C content of 53.1%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values suggested that strain SJY2T represents a new species, for which we propose the name Paenibacillus puerhi sp. nov. with the type strain SJY2T (= CGMCC 1.17156T = KCTC 43242T).

Endophytic Paenibacillus amylolyticus KMCLE06 Extracted Dipicolinic Acid as Antibacterial Agent Derived via Dipicolinic Acid Synthetase Gene.

Bioactive natural compounds play pivotal roles in drug discovery and the emergence of multi-drug resistance pathogens demands the development of better/new drugs. Paenibacillus amylolyticus KMCLE06 endophytic bacterium isolated from the medicinal plant Coix lachryma-jobi were analyzed for the potential bioactive secondary metabolite compounds and its gene responsible within polyketide synthases (PKS) clusters. Ethyl acetate extraction of P. amylolyticus KMCLE06 showed significant antibacterial activity which was further processed to partial purification and characterization for bioactive compound. The foremost bioactive component in extraction was found to be dipicolinic acid (DPA). The antibacterial activity showed remarkable activity compared to the commercial standard DPA against both gram-positive and gram-negative pathogens. The MIC and MBC concentrations for partially purified extracted DPA ranged from 62.5 to 125 µg/ml and MBC from 208 to 250 µg/ml, respectively. Sequence analysis of gene amplified using degenerative primer, amplified 543 bp DNA region, revealing conserved putative open reading frame for dipicolinic acid synthetase (DpsA) key gene to produce DPA in most endospore forming bacteria. A search in the structural database for DpsA revealed significant homologous match with enoyl reductase one of the PKS type 1 module protein. This emphasizes endophytic P. amylolyticus KMCLE06 bacteria has presence of spoVF operon producing DPA via dipicolinic acid synthetase and lacks the polyketide synthase type 1 module cluster gene in its genome. And the bioactive compound DPA extracted acts as a stable remarkable antibacterial agent which can be potent compound for multi-resistance pathogens.

Paenidigyamycin A, Potent Antiparasitic Imidazole Alkaloid from the Ghanaian Paenibacillus sp. DE2SH.

A new alkaloid paenidigyamycin A (1) was obtained from the novel Ghanaian Paenibacillus sp. isolated from the mangrove rhizosphere soils of the Pterocarpus santalinoides tree growing in the wetlands of the Digya National Park, Ghana. Compound 1 was isolated on HPLC at tR = 37.0 min and its structure determined by MS, 1D, and 2D-NMR data. When tested against L. major, 1 (IC50 0.75 µM) was just as effective as amphotericin B (IC50 0.31 µM). Against L. donovani, 1 (IC50 7.02 µM) was twenty-two times less active than amphotericin B (IC50 0.32 µM), reinforcing the unique effectiveness of 1 against L. major. For T. brucei brucei, 1 (IC50 0.78 µM) was ten times more active than the laboratory standard Coptis japonica (IC50 8.20 µM). The IC50 of 9.08 µM for 1 against P. falciparum 3d7 compared to artesunate (IC50 36 nM) was not strong, but this result suggests the possibility of using the paenidigyamycin scaffold for the development of potent antimalarial drugs. Against cercariae, 1 showed high anticercaricidal activity compared to artesunate. The minimal lethal concentration (MLC) and minimal effective concentration (MEC) of the compound were 25 and 6.25 µM, respectively, while artesunate was needed in higher quantities to produce such results. However, 1 (IC50 > 100 µM) was not active against T. mobilensis.

Bacterial Inoculant Treatment of Bermudagrass Alters Ovipositional Behavior, Larval and Pupal Weights of the Fall Armyworm (Lepidoptera: Noctuidae).

Nonpathogenic soil bacteria can colonize the rhizosphere and induce unique plant phenotypes that may influence plant-insect interactions. However, few studies have considered the influences of bacteria-plant interactions on insect feeding and oviposition. The objective of this study was to determine how rhizobacterial inoculation of bermudagrass affects larval development and ovipositional behaviors of the fall armyworm (Spodoptera frugiperda J.E. Smith). Eight blends of rhizobacteria known to induce root or shoot growth in grasses were applied weekly to hybrid bermudagrass for 5 wk. Oviposition was evaluated in two no-choice trials with bacteria-treated, fertilized, or nontreated grass. Grass blades from these treatments were extracted in polar and nonpolar solvents and assayed for oviposition responses. Another experiment compared the development of fall armyworm larvae on bermudagrass treated with each of the eight rhizobacterial blends for 5 wk to larvae fed nontreated bermudagrass. Females deposited more eggs on nontreated and fertilized grass and ≤34% of eggs on grass treated with rhizobacterial blends. Moths exposed to polar and nonpolar extracts were unable to reproduce these results. Larval and pupal weights at days 10 and 12 and the number of adults to eclose were lower for larvae fed some, but not all, bacteria-treated bermudagrass relative to controls. This is one of the few studies to investigate plant-microbe-insect interactions in an economically important system. Although the effects noted with fall armyworm are limited, induced changes in roots also reported for these bacteria may have greater utility than foliar changes for mediating interactions with biotic or abiotic stresses.

Structural insight into a novel neutral metalloproteinase from Paenibacillus spp. BD3526: Implications for mechanisms of rapid inactivation and calcium-dependent stability.

The proteinase with milk-clotting activity (MCA) from Paenibacillus spp. BD3526 is characterized as a neutral metalloproteinase of 35kDa. However, the rapid reduction of its MCA during separation and purification leads to low enzyme recovery. The effects of metal ions, inactivation kinetics, and concentration of calcium on the enzymatic activities and thermal stability of the BD3526 metalloproteinase were investigated. In the absence of calcium, the residual activities of the BD3526 metalloproteinase sharply declined during the first three hours, and continued to slowly decrease thereafter. The activities were well fitted by a double-exponential decay model. The inactivation rates were significantly inhibited by calcium and the residual enzyme activities were maintained at more than 80% for 30d at room temperature with 50-100mM calcium. An intermolecular autoproteolytic mechanism was responsible for BD3526 metalloproteinase inactivation. The target protein band with MCA remained largely undegraded in the enzyme solution that was supplemented with 100mM of calcium, but gradually diminished over time in the absence of calcium. N-terminal amino acid sequencing showed that cleavage at the His252-Ala253 peptide bond facilitated the conversion of the zymogen into the active enzyme. Sequence alignment revealed the presence of two highly conserved motifs, HEXXH and GXXNEXXSD, indicating that the enzyme belonged to the metalloproteinase family M4, also known as thermolysin-like proteinases (TLPs). Further structural analysis showed that the observed calcium-dependent stability of the BD3526 TLP may be attributable to a partly degenerated calcium-binding site, Ca1-2, and a mutant calcium-binding site, Ca3.

Lipopeptides from Bacillus and Paenibacillus spp.: A Gold Mine of Antibiotic Candidates.

The emergence of multidrug-resistant bacteria has placed a strain on health care systems and highlighted the need for new classes of antibiotics. Bacterial lipopeptides are secondary metabolites, generally produced by nonribosomal peptide synthetases that often exhibit broad-spectrum antimicrobial activity. Only two new structural types of antibiotics have entered the market in the last 40 years, linezolid and the bacterial lipopeptide daptomycin. A wide variety of bacteria produce lipopeptides, however Bacillus and Paenibacillus spp. in particular have yielded several potent antimicrobial lipopeptides. Many of the lipopeptides produced by these bacteria have been known for decades and represent a potential gold mine of antibiotic candidates. This list includes the polymyxins, octapeptins, polypeptins, iturins, surfactins, fengycins, fusaricidins, and tridecaptins, as well as some novel examples, including the kurstakins. These lipopeptides have a wide variety of activities, ranging from antibacterial and antifungal, to anticancer and antiviral. This review presents a reasonably comprehensive list of each class of lipopeptide and their known homologues. Emphasis has been placed on their antimicrobial activities, as well other potential applications for this interesting class of substances.

Production of Ginsenoside F2 by Using Lactococcus lactis with Enhanced Expression of ß-Glucosidase Gene from Paenibacillus mucilaginosus.

This study aimed to produce a pharmacologically active minor ginsenoside F2 from the major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing a heterologous ß-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived from Paenibacillus mucilaginosus and synthesized after codon optimization, and the two genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization resulted in reduction of unfavorable codons by 50% and a considerable increase in the expression levels (total activities) of ß-glucosidases (0.002 unit/mL, unoptimized; 0.022 unit/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L. lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng extract. Utilization of this microbial cell to produce F2 may provide an alternative method to increase the health benefits of Panax ginseng.

Paenibacillus ginsengiterrae sp. nov., a ginsenoside-hydrolyzing bacteria isolated from soil of ginseng field.

A novel bacterial strain DCY89(T) was isolated from soil sample of ginseng field and was characterized using a polyphasic approach. Cells were Gram-reaction-positive, rod-shaped, spore-forming and motile with flagella. The strain was aerobic, esculin and starch positive, catalase- and oxidase-negative, optimum growth temperature, and pH were 25-30 °C and 6.0-7.5, respectively. On the basis of 16S rRNA gene sequence analysis, strain DCY89(T) was shown to belong to the genus Paenibacillus and the closest phylogenetic relatives were Paenibacillus cellulosilyticus KACC 14175(T) (98.2%), Paenibacillus kobensis KACC 15273(T) (98.1%), Paenibacillus xylaniclasticus KCTC 13719(T) (96.9%), and Paenibacillus curdlanolyticus KCTC 3759(T) (96.64%). The DNA G+C content was 52.5 mol%, and the predominant respiratory quinone was MK-7. The major fatty acids were iso-C15:0, iso-C16:0, and anteiso-C15:0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The results of the genotypic analysis in combination with chemotaxonomic and physiological data demonstrated that DCY89(T) represented a novel species within the genus Paenibacillus, for which we propose the name Paenibacillus ginsengiterrae. The type strain is DCY89(T) (JCM 19887(T) = KCTC 33430(T)).

Profiling the main cell wall polysaccharides of grapevine leaves using high-throughput and fractionation methods.

Vitis species include Vitis vinifera, the domesticated grapevine, used for wine and grape agricultural production and considered the world's most important fruit crop. A cell wall preparation, isolated from fully expanded photosynthetically active leaves, was fractionated via chemical and enzymatic reagents; and the various extracts obtained were assayed using high-throughput cell wall profiling tools according to a previously optimized and validated workflow. The bulk of the homogalacturonan-rich pectin present was efficiently extracted using CDTA treatment, whereas over half of the grapevine leaf cell wall consisted of vascular veins, comprised of xylans and cellulose. The main hemicellulose component was found to be xyloglucan and an enzymatic oligosaccharide fingerprinting approach was used to analyze the grapevine leaf xyloglucan fraction. When Paenibacillus sp. xyloglucanase was applied the main subunits released were XXFG and XLFG; whereas the less-specific Trichoderma reesei EGII was also able to release the XXXG motif as well as other oligomers likely of mannan and xylan origin. This latter enzyme would thus be useful to screen for xyloglucan, xylan and mannan-linked cell wall alterations in laboratory and field grapevine populations. This methodology is well-suited for high-throughput cell wall profiling of grapevine mutant and transgenic plants for investigating the range of biological processes, specifically plant disease studies and plant-pathogen interactions, where the cell wall plays a crucial role.

Alkalistable xylanase production by alkalitolerant Paenibacillus montaniterrae RMV1 isolated from red mud.

The alkalitolerant and xylanolytic bacterial strain (RMV1) isolated from red mud pond was identified as Paenibacillus montaniterrae based on both biochemical and 16S rDNA sequence analysis. The RMV1 bacterial isolate produced alkalistable and thermostable endoxylanase active over a broad range of pH (4.0-11.0) and temperature (20-100 °C), with optima at 50 °C and pH 9.0 with a T1/2 of 6.7 hours at 50 °C. This is the first report on the isolation of P. montaniterrae from bauxite residue with quite a high xylanase producing ability.

A new antimicrobial and anticancer peptide producing by the marine deep sediment strain "Paenibacillus profundus" sp. nov. Sl 79.

A new linear glyceryl acid derived heptapeptide (1), together with known isocoumarin antibiotic, Y-05460M-A (2), were isolated from the culture of the deep sea sediment strain SI 79 classified as "Paenibacillus profundus" sp. nov. Their structures were determined by 1D- and 2D- NMR techniques and ESI-MS/MS experiments. HPLC analysis of the Marfey derivatives in comparison to their analogs of authentic amino acids revealed that all amino acids in peptide 1, with an exception of Val, have the D-configuration. The compound 1 showed inhibitory activity against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecium as well as cytotoxic and moderate colony growth inhibitory activity against SK-MEL-28 cell line.

Preparation, antioxidant and antitumor activities in vitro of different derivatives of levan from endophytic bacterium Paenibacillus polymyxa EJS-3.

A levan-type exopolysaccharide (EPS) from Paenibacillus polymyxa EJS-3 was successfully acetylated, phosphorylated and benzylated, respectively, affording its derivatives of acetylated levan (AL), phosphorylated levan (PL) and benzylated levan (BL). Then, the antioxidant and antitumor activities in vitro of the natural polysaccharide and its derivatives were determined. As results, AL, BL and PL all exhibited higher reducing power, scavenging activity against superoxide radical and scavenging activity on hydroxyl radical than the natural polysaccharide, EPS-1. In addition, AL, BL and PL also exhibited higher antiproliferative activity against human gastric cancer BGC-823 cells in vitro than EPS-1. The enhanced activities of the derivatives were probably due to the introduction of acetyl, benzyl, or phosphoryl groups into EPS-1 molecules that increased electron-donating ability and affinity with the receptors on immune cells. The results suggested that the derivatives could be explored as promising antioxidant and antitumor agents.