Bacillus subtilis

Powdered grape seeds and bagasse were extracted with petroleum ether to remove fatty material and then re-extracted with different solvent mixtures to determine their total phenolics and antibacterial activities. In the grape seeds extracts the content of total phenolic compounds was found to be 627.98 mg gallic acid equivalent (GAE)/g with acetone:water:acetic acid (90:9.5:0.5) and 667.87 mg GAE/g with ethyl acetate:methanol:water (60:30:10). The bagasse extracts contained 45.44 mg GAE/g and 29.55 mg GAE/g with ethyl acetate:methanol:water (60:30:10) and ethanol:water (95:5), respectively. These extracts at 1%, 2%, 4% and 20% concentrations were tested for their antibacterial effects by using the paper disc diffusion method against some food spoilage and pathogenic bacteria including Aeromonas hydrophila ATCC 7965, Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3,

In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the sporeforming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus.

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of &quot;empty&quot; small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

With growing concerns over multidrug resistance microorganisms, particularly strains of bacteria and fungi, evolving to become resistant to the antimicrobial agents used against them, the identification of new molecular targets becomes paramount for novel treatment options. Recently, the use of new treatments containing multiple active ingredients has been shown to increase the effectiveness of existing molecules for some infections, often with these added compounds enabling the transport of a toxic molecule into the infecting species. Flavonoids are among the most abundant plant secondary metabolites and have been shown to have natural abilities as microbial deterrents and anti-infection agents in plants. Combining these ideas we first sought to investigate the potency of natural flavonoids in the presence of efflux pump inhibitors to limit Escherichia coli growth. Then we used the natural flavonoid scaffold to synthesize non-natural flavanone molecules and further evaluate their antimicrobial efficacy on Escherichia coli, Bacillus subtilis and the fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus. Of those screened, we identified the synthetic molecule 4chloro-flavanone as the most potent antimicrobial compound with a MIC value of 70 mg/mL in E. coli when combined with the inhibitor Phe-Arg-ß-naphthylamide, and MICs of 30 mg/mL in S. cerevesiae and 30 mg/mL in C. neoformans when used alone. Through this study we have demonstrated that combinatorial synthesis of non-natural flavonones can identify novel antimicrobial agents with activity against bacteria and fungi but with minimal toxicity to human cells.

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G + -bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64-512 µg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 µg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M.

Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5. D

One concern with current techniques of UV reactor validation is that they provide only a measure of the mean UV fluence. In this research, the actual fluence distribution of a UV reactor is measured through the use of photochemically active fluorescent microspheres. ...

Bacillomycin Lc, a newantifungal antibiotic of the iturin class, was isolated from a strain of Bacillus subtilis as a set of five congeners. The structure as determined by chemical and spectrometric analyses has been shown to differ from that of bacillomycin L by sequence changes from aspartate-1 to asparagine-1 and from glutamine-5 to glutamate-5. The five congeners differ from each other only in the structure of the aliphatic side chain of the constituent /?-amino acid. The hydrophobicity of the /?-amino acid affects the antifungal activity of the congener, as activity increased in the order of increased congener retention on a reversed-phase HPLCcolumn.

Although several strains ofB. subtiliswith antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a nativeB. subtilisstrain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis21) demonstratedin vitroantagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected withRhizoctonia solaniandFusarium verticillioidesand treated withB. subtilis21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition,B. subtilis21 showed activity against pathogenic bacteria. Secreted proteins byB. subtilis21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then,B. subtilis21 could potentially be used to control phyt...

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (b ⁄ a) 8 -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes.

ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to Pglycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342. D

The polymerase chain reaction (PCR) catalyzed by Taq DNA polymerase has been used to amplify genomic and plasmid DNA sequences. In all reported cases, the template has been extensively purified. We show here that genomic and plasmid DNA sequences can be amplified by the PCR directly from single colonies o f both Escherichia coli and Bacillus subtilis without any purification of the template. This technique makes the use of the PCR even more applicable in the automation processes, screening and diagnosis.

Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides + 37 and + 159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/Grich sequences that symmetrically blank a short inverted repeat.

Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/ MS) analysis.

Acacia nilotica subsp. kraussiana was reported in African traditional medicine for the treatment of various ailments. Isolation of an active compound in this study from the bark extract may lead to the validation of its efficiency as a traditional crude drug.This study aimed to isolate active compound(s) from an ethyl acetate bark extract of Acacia nilotica subsp. kraussiana and to investigate some of its biological activity.The isolation process was carried out using bioassay-guided fractionation. The isolated compound was tested for antibacterial activity using the micro-dilution assay; anti-inflammatory activity using the COX-1 and COX-2 assays and investigated for inhibitory effect against acetylcholinesterase using the microplate assay.A new bioactive compound was isolated and identified as a cassane diterpene, niloticane. Niloticane showed antibacterial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus with MIC values of 4 and 8 μg/mL, respectively. With Gram-negative bacteria, niloticane showed weak activity. MIC values obtained were 16 and 33 μg/mL against Klebsiella pneumonia and Escherichia coli, respectively. In the cyclooxygenase test, niloticane possessed activity with IC50 values of 28 and 210 μM against COX-1 and COX-2, respectively. IC50 values observed with indomethacin (positive control) were 3.6 μM for COX-1 and 189 μM for COX-2. In the acetylcholinesterase test, niloticane showed anti-cholinesterase activity with an IC50 value of 4 μM. IC50 values obtained by the galanthamine (positive control) was 2.0 μM.The results obtained support the traditional uses of the bark of Acacia nilotica subsp. kraussiana in African traditional medicine for the treatment of some ailments that relate to microbial diseases, inflammation and central nervous system disorders.

Due to their recognised properties of biocompatibility, biodegradability and sustainability, chitosan nanocarriers have been successfully used as new delivery systems. In this work, nanoparticles combining chitosan and lignosulfonates were developed for the first time for cosmetic and biomedical applications. The ability of lignosulfonates to act as a counter polyion for stabilisation of chitosan particles, generated using high intensity ultrasound, was investigated. Several conditions for particles preparation were tested and optimised and the resulting nanoparticles were comprehensively characterised by measuring particle size, zeta potential and polydispersity index. The pH of chitosan solution, sonication time and the presence of an adequate surfactant, poloxamer 407, were determinant factors on the development of smaller particles with low polydispersity index (an average particle size of 230 nm was obtained at pH 5 after 8 min of sonication). The beneficial effects of lignosulfonates complex on chitosan nanoparticles were further characterised. Greater stability to lysozyme degradation, biocompatibility with human cells and antimicrobial activity was found upon lignosulfonates incorporation into chitosan nanoparticles. Furthermore, these particles were able to incorporate a hydrophilic model protein -RNase A. A burst release was observed when nanoparticles were loaded with low amount of protein while with high protein content, a sustained release was found, suggesting that the protein cargo maybe loaded both at the surface as in the bulk of the particle, depending on the concentration of drug incorporated.

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.

Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.

Global food shortage is mainly due to the post-harvest spoilage of food, rather than its under-production, especially in the sub-Saharan African countries. Rapid detection tools for the spoilage of fruit such as banana (Musa acuminata) would be a great resource in alleviating losses during post-harvest storage. In this study, silver-based colloidal nanoparticle (AgNPs) solution was synthesized using culture supernatant or wet biomass (cell pellets) of Bacillus subtilis. The synthesized silver nanoparticles solution was confirmed using UV-Vis Spectrophotometer and further characterized using Scanning Electron Microscope (SEM), X-ray Diffractometer (XRD) and Fourier Transmission Infrared (FT-IR), and then evaluated as a colorimetric sensor for the volatile compound released during the deterioration of Musa acuminata. The original reddish brown colour of the silver nanoparticle solution changed to light brown by the end of the fourth day and finally turned transparent on the tenth day. Simultaneous analysis carried out by UV-Vis spectroscopy corroborates our investigation of AgNPs as a colorimetric sensor displaying selectivity and specificity for 1,2-Benzenedicarboxylic acid, bis (2-methyl propyl) ester which was released during the deterioration of Musa acuminata. The use of colorimetric sensor as conducted in this study will help in easy detection for control of crop deterioration.

The cytoskeletal protein, FtsZ plays a pivotal role in prokaryotic cell division and is present in majority of the bacterial species. In recent years, inhibitors of FtsZ have been identified that may function as lead compounds for the development of novel antimicrobials. It has been found that curcumin, the main bioactive component of Curcuma longa, inhibits Bacillus subtilis and Escherichia coli growth by inhibiting FtsZ assembly. Though it is experimentally established that curcumin inhibits FtsZ polymerization, the binding site of curcumin in FtsZ is not known. In this study, interaction of curcumin with catalytic core domain of E. coli and B. subtilis FtsZ was investigated using computational docking.

The leuB gene codes for 3-lsopropylmalate dehydrogenase of the leucjne biosynthetic pathway in an extreme thermophile. Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucJne was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti-and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP.

Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in th...

PLATE 1. Treatment with buffered oso4 showing membrane ruptured in one place (x 20,000). PLATE 2. Treatment with 0.06 g/1 cetrimide. Membranes of varying sizes and degrees of disorganization together with a few small granules (x 8000). Plates 1-6 show h'. coli sphoroplasts shadowed with gold and palladium after troatment with Bact. f. p. 452. antiseptics or fixative.

Two field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21) on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3–13.5% for cucumber plants treated with bioorganic fertilizer, while it was 30.3–51% in controls (only with organic fertilizer). Higher yields and lower disease incidences were observed in the dry season when compared with the wet season for both types of organic fertilizer treatments. Biolog analysis showed a significant change in soil bacterial composition and activity after bioorganic fertilizer application. The numbers of colony-forming units of F. oxysporum f. sp. Cucumerinum J. H. Owen for bioorganic-fertilizer-treated soils were significantly decreased compared with control. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had low carbon and oxygen than the living mycelia. Reverse-phase high-pressure liquid chromatography and mass spectroscopic analysis showed that the antagonistic bacteria produced the antifungal compounds fusaricidin A, B, C, and D with molecular weights of 883.5, 897.5, 947.5, and 961.5 Da, respectively. The application of bioorganic fertilizer has a great potential for the control of F. oxysporum wilt disease in cucumber plants.

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10 6 , 10 9 and 10 12 CFU ml À1 E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.

Chlorine Ultraviolet light Ozone A B S T R A C T Veterinary antibiotics are widely used at concentrated animal feeding operations (CAFOs) to prevent disease and promote growth of livestock. However, the majority of antibiotics are excreted from animals in urine, feces, and manure. Consequently, the lagoons used to store these wastes can act as reservoirs of antibiotics and antibiotic-resistant bacteria.

Thin-layer chromatography of ethanolic extract of propolis (EEP) from the continental and Adriatic regions of Croatia showed that 72.2% of propolis samples contain galangin, 88.8% of samples contain kaempferol, naringenin and apigenin and 66.6% of samples contain caffeic acid. Caffeic acid, pinocembrin, galangin, chrysin and naringenin were analyzed by HPLC. In all samples, pinocembrin was the dominant flavonoid. In samples from the Adriatic region, concentration of pinocembrin ranged from 0.03 to 6.14% (x = 2.87%) and in the continental region samples from 0 to 4.74% (x = 2.84%). Chrysin was found in all propolis samples in a concentration ranging from 0.22 to 5.32% (x = 1.86%) in the continental region samples and from 0.03 to 3.64% (x = 1.96%) in samples from the Adriatic region. Chrysin was followed by naringenin, ranging from 0 to 1.14% (x = 0.42%) in samples from the Adriatic region and from 0.22 to 2.41% (x = 0.60%) in the continental region samples. Concentration of caffeic ...

The goal of this paper is to explore the possibility to detect and identify bacteria by sensing their odor via fluctuation-enhanced sensing with commercial Taguchi sensors. The fluctuations of the electrical resistance during exposure to different bacterial odors, Escherichia coli and anthrax-surrogate Bacillus subtilis, have been measured and analyzed. In the present study, the simplest method, the measurement and analysis of power density spectra was used. The sensors were run in the normal heated and the sampling-and-hold working modes, respectively. The results indicate that Taguchi sensors used in these fluctuation-enhanced modes are effective tools of bacterium detection and identification even when they are utilizing only the power density spectrum of the stochastic sensor signal.

During 2005 growing season, field experiments were conducted in two commercial raspberry fields to compare effectiveness of some fungicides and biofungicides in Botrytis cinerea control. The experiments consisted of four fungicide and two biofungicide treatments and untreated control, in randomized block design with four replicates per treatment. The fungicides: fenhxamid, vinclozolin, benomyl, and pyrimethanil were applied twice. The biofungicides Polyversum

The potential eco-toxicity of nanosized titanium dioxide (TiO2), silicon dioxide (SiO2), and zinc oxide (ZnO) water suspensions was investigated using Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as test organisms. These three photosensitive nanomaterials were harmful to varying degrees, with antibacterial activity increasing with particle concentration. Antibacterial activity generally increased from SiO2 to TiO2 to ZnO, and B. subtilis was most susceptible to their effects. Advertised nanoparticle size did not correspond to true particle size. Apparently, aggregation produced similarly sized particles that had similar antibacterial activity at a given concentration. The presence of light was a significant factor under most conditions tested, presumably due to its role in promoting generation of reactive oxygen species (ROS). However, bacterial growth inhibition was also observed under dark conditions, indicating that undetermined mechanisms additional to photocatalytic ROS production were responsible for toxicity. These results highlight the need for caution during the use and disposal of such manufactured nanomaterials to prevent unintended environmental impacts, as well as the importance of further research on the mechanisms and factors that increase toxicity to enhance risk management.

Aim: The aim of the study was to determine the potentials and effects of Bacillus subtilis and lactic acid bacteria (LAB) as probiotics on broiler growth, health, and Salmonella infection. Materials and Methods: To evaluate the inoculum size applicable for broilers, 1-day-old broilers were orally fed fresh cultures of single strains and a B. subtilis KKU213/Pediococcus pentosaceus NP6 mixture at 108 and 1012 colony-forming unit (CFUs)/mL/chick. The body weight gain (BWG), Salmonella contamination level and total Bacillus and LAB abundances in the crop and intestine were measured. Subsequently, 1-day-old broilers were orally fed of KKU213, CH403, and Pediococcus acidilactici SH8 at 1010 CFUs/mL, followed by inulin. After 35 days, the BWG, Bacillus and LAB abundances in the cecum, blood parameters, and KKU213 colonization were assessed. Results: The broilers fed single strains or KKU213+NP6 exhibited a higher BWG and a higher crop LAB abundance than the controls (p&lt;0.05). Probiotic...

The antimicrobial properties of the crude and pure extracts of Nerium oleander were investigated against three Gram-ve bacteria (Escherichia coli, Pseudomonas aeruginosa and Salmonella enteritidis), three Gram +ve bacteria (Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus) and six fungal species (Aspergillus flavus, A. fumigatus, A. niger, Fusarium moniliforme, Penicillium expansum and Rhizopus oryzae). The crude extract of N. oleander was more effective than purified compound. The maximum antimicrobial activities (15 and 20 mm inhibition zone) was exhibited by the crude extract of N. oleander against the most sensitive isolates (B. subtilis and A. flavus), respectively, while all pure constituents showed trace to no antimicrobial activity against the tested microorganisms. The lowest value (25 μg/ml) of minimum inhibitory concentration of the active crude extract of N. oleander was revealed with A. flavus.

Ten new azo Schiff bases 5a-h and 7a-b were prepared in excellent yields via the condensation of different aromatic amines and a new azoaldehyde, 2-hydroxy-3methoxy-5-(4-methoxyphenylazo)benzaldehyde (4) by two different methods. All new compounds were tested against five microorganisms: Staphylococcus aureus (Gram positive and methicillin resistant), Bacillus subtilis (Gram positive), Kelebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli (all Gram negative). Compounds 4, 5a, 5c, 5d and 5g were moderately active against Staphylococcus aureus and Bacillus subtilis. Compound 7b was highly active against Bacillus subtilis and moderately active against Staphylococcus aureus. Other compounds were inactive against these strains of bacteria. The antifungal activities of these compounds were also tested against eight different fungal species. None of them were active against the fungi species tested.

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo-). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo- mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram-negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide-transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP-binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide-binding protein, which is periplasmically located in Gram-negative species, has a signal sequence characteristic of lipo-proteins with an amino-terminal lipo-amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram-negative bacteria is the recycling of cell-wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signaling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.

& The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w=w) cellulose, 54.4% (w=w) hemicellulose, and 16.9% (w=w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g=L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w=v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g=L. Employing 1% (w=v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g=L and 1.52 g=L, respectively, were obtained. The SSF experiments performed with 5% (w=v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g=L using the shake flask and 12.33 g=L at the bioreactor level. From the bioreactor, 94.4% (v=v) ethanol was recovered by rotary evaporator with 21% purification efficiency.

The process of cell division has been intensively studied at the molecular level for decades but some basic questions remain unanswered. The mechanisms of cell division are probably best characterized in the rod-shaped bacteria Escherichia coli and Bacillus subtilis. Many of the key players are known, but detailed descriptions of the molecular mechanisms which determine where, how and when cells form the division septum are lacking. Different models have been proposed to account for the high precision with which the septum is constructed at the midcell and these models have been evaluated and refined against new data emerging from the fast improving methodologies of cell biology. This review summarizes important advances in our understanding of how the cell positions the division septum, whether it be vegetative or asymmetric. It also describes how the asymmetric septum forms and how this septation event is linked to chromosome segregation and subsequent asymmetric gene expression during spore formation in B. subtilis.

Aims:  To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates.Methods and Results:  The spores of B. subtilis were prepared at 37°C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid-soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed ∼threefold more rapidly at 90°C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross-linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso- to iso-fatty acids.Conclusions:  The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross-linking of a coat protein in spores made in liquid and on plates.Significance and Impact of the Study:  This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.

Heliotropamide (1), a new alkaloid with a novel oxopyrrolidine-3-carboxamide central moiety, has been isolated as the major product of the dicholoromethane extract of Heliotropium ovalifolium aerial parts. Its structure was elucidated by spectrometric methods including ESI-HR, EI, D/CI mass spectrometry, 1 H, 13 C, and 2D NMR experiments, and chemical derivatization. Neither heliotropamide nor its acetylated derivative (1a) showed any antifungal activity against Cladosporium cucumerinum and Candida albicans, antibacterial activity against Bacillus subtilis, radical-scavenging properties in the DPPH test, or inhibitory potential toward acetylcholinesterase.

Artemisia annua L. (Asteraceae) is an important medicinal plant whose secondary metabolite artemisinin is used for the treatment of cerebral malaria. A study was undertaken to determine the effect of arbuscular mycorrhizal (AM) fungi, Glomus mosseae, Glomus aggregatum, Glomus fasciculatum, Glomus intraradices and two free living nitrogen fixing bacteria (NFB) (identified as Bacillus subtilis and Stenotrophomonas spp. through 16S rRNA gene sequence analysis), inoculated alone or in combinations on the biomass, nutrient uptake, and content of artemisinin in A. annua under glass house conditions. Various parameters like plant height, total plant biomass, NPK content, leaf yield, mycorrhizal colonization, bacterial population in rhizosphere, phosphatase activity and artemisinin content were determined and found to vary with different treatments. Among all the treatments, plants inoculated with G. mosseae + B. subtilis performed better than any other treatment or uninoculated control plants. The results of the experiment clearly indicated the compatibility and synergy between G. mosseae and B. subtilis and suggested the use of this microbial consortium in A. annua for enhancing growth, biomass yield, and the content and yield of artemisinin.

This study investigated the effectiveness of detergent and aqueous solutions of 2% chlorhexidine digluconate in decontaminating gutta-percha cones (gpc) contaminated with bacteria, yeast, or bacterial spores. Guttapercha cones were contaminated with 10 7 -10 8 colony-forming units per milliliter (cfu/ml) of the following test organisms: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, or Candida albicans. Spores of Bacillus subtilis were also tested. Contaminated gpc were treated with the chlorhexidine solutions for 1, 5, 10, or 15 min. Each cone was then transferred to a tube containing saline and the micoorganisms were recovered after homogenization for cfu determination. Both detergent and aqueous chlorhexidine solutions were effective in eliminating S. aureus, E. faecalis, and C. albicans cells adhered on the surface of gpc within 1 min of exposure. E. coli was eliminated in 5 min with detergent solution. The Bacillus subtilis spores were eliminated by chlorhexidine solutions within 5 min. The results of this study demonstrated that both aqueous and detergent solutions of 2% chlorhexidine digluconate were effective in decontaminating gpc within 5 minutes of exposure.