Proteome analysis of Paenibacillus polymyxa E681 affected by barley

Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the train was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1000 spots in the cell and 1100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-alanine ligase A, isopentenyldiphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.     

Functional identification and expression of indole-3-pyruvate decarboxylase from Paenibacillus polymyxa E681

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylaselike proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5alpha. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.     

Identification of a new gene PA5017 involved in flagella-mediated motility, chemotaxis and biofilm formation in Pseudomonas aeruginosa

Flagella-mediated motility is recognized as one of the major factors contributing to virulence in Pseudomonas aeruginosa. During a screening of a mini-Mu transposon mutant library of P. aeruginosa PA68, a mutant partially deficient in swimming and swarming motility was identified in a new locus that encodes a predicted protein of unknown function annotated PA5017 in the P. aeruginosa PAO1 genome sequence. Chemotaxis plate assay indicated that inactivation of the PA5017 gene led to a decreased chemotactic response. Complementation of the PA5017 mutant with the wild-type PA5017 gene restored normal motility and chemotaxis phenotype. A promoter-lacZ reporter activity assay of the cheYZAB operon from chemotaxis gene cluster 1 showed that there was almost a twofold difference in expression levels of the wild-type PA68 and the PA5017 mutant. This suggested that the PA5017 affected expression of the cheYZAB operon negatively. Further study showed that inactivation of the PA5017 gene in PA68 led to increased biofilm formation in a static system and to the formation of a heterogeneous biofilm in a flow-chamber system. These results suggested that PA5017 possibly affected flagellum-dependent motility and in turn biofilm formation via the chemotaxis signal transduction pathway.     

Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates

This study aimed to identify bacteria involved in Trichodermaharzianum inhibition while promoting Pleurotus ostreatus defences in order to favour cultivation-substrate selectivity for mushroom production. PCR-DGGE profiles of total DNA from wheat-straw substrate showed weak differences between bacterial communities from substrate inoculated with P. ostreatus with or without T. harzianum. The major cultivable bacteria were isolated from three batches of wheat-straw-based cultivation substrates showing an efficient selectivity. They were screened for their ability to inhibit T.harzianum. By using specific media for bacterial isolation and by sequencing certain 16S-rDNA, we observed that Bacillus spp. were the main inhibitors. Among them, a dominant species was identified as Paenibacillus polymyxa. This species was co-cultivated on agar media with P. ostreatus. The measurement of laccase activities from culture plugs indicated that P. polymyxa induced increases in enzyme activities. Bacillus spp. and specifically P. polymyxa from cultivation substrates are implicated in their selectivity by both inhibiting the growth of T.harzianum and stimulating defences of the mushroom P. ostreatus through the induction of laccases. The management of microbial communities during P.ostreatus cultivation-substrate preparation in order to favour P. polymyxa and other Bacillus spp. growth, can be a way to optimize the development of P. ostreatus for mushroom production or other environmental uses of this fungus.     

Active inclusion body formation using Paenibacillus polymyxa PoxB as a fusion partner in Escherichia coli

Overexpression of Paenibacillus polymyxa PoxB in Escherichia coli induced the formation of inclusion bodies. An enzyme assay showed that the inclusion bodies exhibited PoxB activity, indicating that they were biologically active. Fusion of GFP and Bacillus subtilis AmyE to the C-terminus of the PoxB also induced the formation of biologically active aggregates when they were overexpressed in E. coli. Therefore, P. polymyxa PoxB can be used as a fusion partner to promote the formation of active inclusion bodies in E. coli.     

Improvement in bioavailability of tricalcium phosphate to Cymbopogon martinii var. motia by rhizobacteria, AMF and Azospirillum inoculation

The interactive effects of phosphate solubilizing bacteria, N2 fixing bacteria and arbuscular mycorrhizal fungi (AMF) were studied in a low phosphate alkaline soil amended with tricalcium insoluble source of inorganic phosphate on the growth of an aromatic grass palmarosa (Cymbopogon martinii). The microbial inocula consisted of the AM fungus Glomus aggregatum, phosphate solubilizing rhizobacteria Bacillus polymyxa and N2 fixing bacteria Azospirillum brasilense. These rhizobacteria behaved as "mycorrhiza helper" and enhanced root colonization by G. aggregatum in presence of tricalcium phosphate at the rate of 200 mg kg(-1) soil (P1 level). Dual inoculation of G. aggregatum and B. polymyxa yielded 21.5 g plant dry weight (biomass), while it was 21.7 g in B. polymyxa and A. brasilense inoculated plants as compared to 14.9 g of control at the same level. Phosphate content was maximum (0.167%) in the combined treatment of G. aggregatum, B. polymyxa and A. brasilense at P1 level, however acid phosphatase activity was recorded to be 4.75 pmol mg(-1) min(-1) in G. aggregatum, B. polymyxa and A. brasilense treatment at P0 level. This study indicates that all microbes inoculated together help in the uptake of tricalcium phosphate which is otherwise not used by the plants and their addition at 200 mg kg(-1) of soil gave higher productivity to palmarosa plants.     

Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia

The taxonomy of the asymmetric (predominantly terverticillate) penicillia is based on morphological differences that leave identification difficult. The application of physiological criteria facilitated the identification of the common asymmetric penicillia investigated. Changes in the placement of some strains of these penicillia made the connection to mycotoxin-producing ability clearer. The classical criterion of conidium color was deemphasized and replaced by the following criteria: (i) growth on nitrite-sucrose agar and (ii) growth and acid (and subsequent base) production on creatine-sucrose agar (containing bromocresol purple). Other criteria used or developed were: (iii) growth on sorbic acid plus benzoic acid agar (50 + 50 ppm, pH 3.8), (iv) growth on an agar containing 1,000 ppm propionic acid (pH 3.8), (v) growth on an agar containing 0.5% acetic acid, (vi) growth at 37 degrees C, (vii) growth rate on an agar containing 0.1% pentachloronitrobenzene, (viii) production of extracellular tricaproinase, and (ix) fasciculation on a medium containing 10 ppm botran (2,6-dichloro-4-nitroanilin). The pattern of extracellular metabolites after thin-layer chromatography was used as a chemotaxonomic criterion. The species investigated, the number of isolates investigated, and the toxins which some of these isolates produce were: Penicillium roqueforti (18) (patulin), P. citrinum (11) (citrinin), P. patulum (9) (patulin and griseofulvin), P. expansum (patulin and citrinin), P. hirsutum (13), P. brevicompactum (19), and P. chrysogenum (12). Widespread species of the P. cyclopium, P. viridicatum, and P. expansum series of Raper and Thom (A Manual of the Penicillia, 1949) were subdivided into four new groups: "P. crustosum pA" (29) (penitrem A), "P. melanochlorum" (29), "P. cyclopium p" (119) (penicillic acid and infrequently penitrem A), and "P. viridicatum o-c" (43) (ochratoxin A and citrinin). "P. viridicatum o-c" was separated from "P. cyclopium p" due to its ability to grow on nitrite as sole nitrogen source. The species and groups investigated were related to the new taxonomic classification of the genus Penicillium according to Pitt.     

The Effects of Paenibacillus polymyxa E681 on Antifungal and Crack Remediation of Cement Paste

This study investigated the antifungal effects of cement paste containing Paenibacillus polymyxa E681 against Aspergillus niger, a deleterious fungus commonly found in cement buildings and structures. To test the antifungal effects, cement paste containing P. polymyxa E681 was neutralized by CO₂ gas, and the fungal growth inhibition was examined according to the clear zone around the cement specimen. In addition to the antifungal effects of the cement paste added with bacteria, calcium crystal precipitation of P. polymyxa E681 was examined by qualitative and quantitative analyses. The cement paste containing P. polymyxa E681 showed strong antifungal effects but fusA mutant (deficient in fusaricidin synthesis) showed no antifungal activity. Crack sealing of the cement paste treated with P. polymyxa E681 was captured by light microscope showed fungal growth inhibition and crack repairing in cement paste.     

Membrane filter (pore size, 0.22-0.45 micro m; thickness, 150 micro m) passing-through activity of Pseudomonas aeruginosa and other bacterial species with indigenous infiltration ability

Bacteria growing on MF-Millipore filters (thickness, 150 micro m) passed through the underlying membrane by their infiltration activity. Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli passed through a 0.45- micro m pore size filter within 48-96 h. Pseudomonas aeruginosa, Serratia marcescens, and Listeria monocytogenes passed through a 0.3- micro m pore size filter. P. aeruginosa passed through a 0.22- micro m pore size filter. The membranes which allowed passing-through of bacteria showed normal bubble point values in the integrity test. Studies with isogenic S. marcescens mutants indicated that flagellum-dependent motility or surface-active exolipid were important in the passing-through. P. aeruginosa PAO1 C strain defective in twitching motility was unable to pass through the 0.22- micro m filter. Scanning electron microscopy showed bacteria passing-through the 0.22- micro m filter. Millipore membrane filters having well-defined reticulate structures will be useful in the study of infiltration activity of microbes.     

Evidence for a second binding/transport site for chloride in erythrocyte anion transporter AE1 modified at glutamate 681

Transport kinetics have been examined in erythrocyte anion transporter AE1 that has been chemically modified to convert glutamate 681 to an alcohol (E681OH AE1). Outward conductive Cl(-) flux in E681OH AE1 is inhibited by removal of extracellular Cl(-); this effect is the opposite of that in native AE1 and is consistent with coupled electrogenic 2:1 Cl(-)/Cl(-) exchange. A second Cl(-) binding/transport site is also suggested by the characteristics of (35)SO(4)(2-) flux in E681OH AE1: bilateral and cis Cl(-), which are normally inhibitory, accelerate (35)SO(4)(2-) flux. These effects would be expected if Cl(-) binds to a second transport site on SO(4)(2-)-loaded E681OH AE1, thereby allowing Cl(-)/SO(4)(2-) cotransport. Alternatively, the data can be explained without proposing Cl(-)/SO(4)(2-) cotransport if the rate-limiting event for (35)SO(4)(2-)/SO(4)(2-) exchange is external SO(4)(2-) release, and the binding of external Cl(-) accelerates SO(4)(2-) release. With either interpretation, these data indicate that E681OH AE1 has a binding/transport site for Cl(-) that is distinct from the main transport site. The effects of graded modification of E681 or inhibition by H(2)DIDS are consistent with the idea that the new Cl(-) binding site is on the same E681OH-modified subunit of the AE1 dimer as the normal transport site.     

A major decomposition product, citrinin H2, from citrinin on heating with moisture

Citrinin is one of the mycotoxins produced by Penicillium citrinum. We examined the decomposition products after heating citrinin in water at 140 degrees C and isolated a major product, citrinin H2 (3-(3,5-dihydroxy-2-methylphenyl)-2-formyloxy-butane). Citrinin H2 did not show significant cytotoxicity to HeLa cells up to a concentration of 200 microg/ml (% cytotoxicity: 39%) in 63 h of incubation, but citrinin showed severe toxicity at a concentration of 25 microg/ml (% cytotoxicity: 73%). HPLC analysis of citrinin after heating under various conditions indicates that citrinin H2 is mainly yielded from citrinin.     

Studies using an in vitro model show evidence of involvement of epithelial-mesenchymal transition of human endometrial epithelial cells in human embryo implantation

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.     

Pantoea stewartii subsp. stewartii exhibits surface motility, which is a critical aspect of Stewart's wilt disease development on maize

Pantoea stewartii subsp. stewartii is a plant-pathogenic bacterium that causes Stewart's vascular wilt in maize. The organism is taxonomically described as aflagellated and nonmotile. We recently showed that P. stewartii colonizes the xylem of maize as sessile, cell-wall-adherent biofilms. Biofilm formation is a developmental process that generally involves some form of surface motility. For that reason, we reexamined the motility properties of P. stewartii DC283 based on the assumption that the organism requires some form of surface motility for biofilm development. Here, we show that the organism is highly motile on agar surfaces. This motility is flagella dependent, shown by the fact that a fliC mutant, impaired in flagellin subunit synthesis, is nonmotile. Motility also requires the production of stewartan exopolysaccharide. Moreover, surface motility plays a significant role in the colonization of the plant host.     

Natural variation of gliding motility in a centimetre-scale population of Myxococcus xanthus

A major challenge in microbial evolutionary ecology is to understand how fitness-related traits vary in natural populations of microorganisms at defined spatial scales and subsequently to identify the forces that maintain such variation. The Gram-negative soil bacterium Myxococcus xanthus is a model system for the study of gliding motility, which is driven by two complementary motility systems in this species and is central to its social lifestyle. We tested whether the ecological context of a centimetre-scale M. xanthus population allows the coexistence of diverse motility-related phenotypes. Swarming rates among 26 clones isolated at the centimetre scale were found to vary greatly in multiple laboratory environments. This variation appears to be motility-specific, as it is not explained by a correlated variation in intrinsic growth rate. In contrast to the common reference strain DK1622, most isolates swarmed faster on hard agar than on soft agar, highlighting the difficulty of inferring species characteristics from laboratory reference strains. These isolates also varied greatly in swarm morphology and in the effect of nutrient limitation on swarming rate. Our results show that diverse swarming phenotypes can coexist in a small-scale bacterial population.     

Paenibacillus polymyxa invades plant roots and forms biofilms

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.     

Biological control and plant growth promoting capacity of rhizobacteria on pepper under greenhouse and field conditions

Plant growth promoting rhizobacteria Ochrobactrum lupini KUDC1013 and Novosphingobium pentaromativorans KUDC1065 isolated from Dokdo Island, S. Korea are capable of eliciting induced systemic resistance (ISR) in pepper against bacterial spot disease. The present study aimed to determine whether plant growth-promoting rhizobacteria (PGPR) strains including strain KUDC1013, strain KUDC1065, and Paenibacillus polymyxa E681 either singly or in combinations were evaluated to have the capacity for potential biological control and plant growth promotion effect in the field trials. Under greenhouse conditions, the induced systemic resistance (ISR) effect of treatment with strains KUDC1013 and KUDC1065 differed according to pepper growth stages. Drenching of 3-week-old pepper seedlings with the KUDC-1013 strain significantly reduced the disease symptoms. In contrast, treatment with the KUDC1065 strain significantly protected 5-week-old pepper seedlings. Under field conditions, peppers treated with PGPR mixtures containing E681 and KUDC1013, either in a two-way combination, were showed greater effect on plant growth than those treated with an individual treatment. Collectively, the application of mixtures of PGPR strains on pepper might be considered as a potential biological control under greenhouse and field conditions.