Bacillus subtilis para el control del oidio de la calabaza.

2009

Caracterización funcional de las proteínas TasA y TapA en la formación de fibras de tipo amiloide en Bacillus subtilis

Las proteínas amiloides son un grupo heterogéneo de proteínas diferentes en secuencia aminoacídica, pero similares en su estructura cuaternaria: fibras enriquecidas en láminas beta, con gran estabilidad, resistencia y capacidad de unión de colorantes específicos, como el rojo congo o la thioflavina T. Estas proteínas han estado tradicionalmente asociadas a patologías neurodegenerativas en humanos como el Alzheimer o el Parkinson. Sin embargo, los miembros dentro de esta familia están ampliamente distribuidas en la naturaleza, desde bacterias hasta humanos, e intervienen en un amplio rango de funciones biológicas, motivo por el que se han denominado “amiloides funcionales”. En bacterias, los amiloides funcionales son responsables de participar en funciones muy diversas como la interacción célula-célula, con superficies abióticas, y formación de biofilms. En Bacillus subtilis, la proteína amiloide TasA es el componente proteico mayoritario de la matriz extracelular del biofilm de este microorganismo y el principal elemento que constituye las fibras amiloides, mientras que la proteína auxiliar TapA, presente en mucha menor proporción, actúa favoreciendo el ensamblaje de las mismas. Estas actúan como un andamiaje proteico donde se disponen el resto de componentes de la matriz extracelular, lo que confiere a esta estructura una mayor estabilidad y, por consiguiente, proporcionan una mayor robustez al biofilm. En este este trabajo se pretende llevar a cabo el análisis de regiones o dominios tanto de TasA como de TapA importantes para la amiloidogénesis, así como para la funcionalidad de ambas proteínas. Para ello, el estudio se ha enfocado desde un punto de vista multidisciplinar, combinando pruebas clásicas de caracterización de amiloides con técnicas de biología molecular y diversas pruebas biofísicas. Los resultados obtenidos hasta la fecha han demostrado la existencia de pequeñas secuencias, dentro de las proteínas TasA o TapA con capacidad para polimerizar en la forma de fibras, lo que muestra su importancia en el proceso de fibrilación y en la funcionalidad de ambas proteínas. En el caso de la proteína TapA, las regiones analizadas ponen de manifiesto la importancia de su extremo amino-terminal tanto en la funcionalidad de la proteína como en su interacción con TasA.

Respostas fisiológicas de pirarucu ao probiótico Bacillus subtilis (Cohn 1872).

O uso de probióticos na alimentação dos peixes pode estimular o sistema imunológico e agir na prevenção de enfermidades. O presente estudo avaliou a fisiologia de pirarucu Arapaima gigas após alimentação por 30 dias com dietas enriquecidas com probiótico Bacillus subtilis em 3 níveis de inclusão: 0 (Controle); 0,02 (BS0,02%) e 0,05% (BS0,05%) (triplicata), sendo realizada biometria e coleta de amostras de sangue. Os grupos foram comparados entre si (P<0,05). O peso, comprimento e o índice hepatossomático (IHS) dos pirarucus BS0,05% foram menores que os do Controle. As análises hematológicas e bioquímicas indicaram que BS0,02% promoveu aumento do hematócrito e da albumina, com relação aos demais grupos; e diminuição da concentração de hemoglobina corpuscular média (CHCM) e da glicose plasmática com relação ao Controle. A hemoglobina corpuscular média (HCM) e atividade respiratória dos leucócitos do BS0,05% diminuíram com relação ao Controle. BS0,02% apresentaram redução do número de leucócitos, com relação ao Controle, que refletiu na diminuição de monócitos, neutrófilos, LG-PAS e eosinófilos. Assim, os níveis de B. subtilis avaliados não promoveram crescimento ou alterações fisiológicas nos pirarucus, que indiquem melhoria do seu sistema imune.

Controle da ferrugem do cafeeiro com produtos à base de Bacillus subtilis.

1994

Genomic And Chemical Insights Into Biosurfactant Production By The Mangrove-derived Strain Bacillus Safensis Ccma-560.

Many Bacillus species can produce biosurfactant, although most of the studies on lipopeptide production by this genus have been focused on Bacillus subtilis. Surfactants are broadly used in pharmaceutical, food and petroleum industry, and biological surfactant shows some advantages over the chemical surfactants, such as less toxicity, production from renewable, cheaper feedstocks and development of novel recombinant hyperproducer strains. This study is aimed to unveil the biosurfactant metabolic pathway and chemical composition in Bacillus safensis strain CCMA-560. The whole genome of the CCMA-560 strain was previously sequenced, and with the aid of bioinformatics tools, its biosurfactant metabolic pathway was compared to other pathways of closely related species. Fourier transform infrared (FTIR) and high-resolution TOF mass spectrometry (MS) were used to characterize the biosurfactant molecule. B. safensis CCMA-560 metabolic pathway is similar to other Bacillus species; however, some differences in amino acid incorporation were observed, and chemical analyses corroborated the genetic results. The strain CCMA-560 harbours two genes flanked by srfAC and srfAD not present in other Bacillus spp., which can be involved in the production of the analogue gramicidin. FTIR and MS showed that B. safensis CCMA-560 produces a mixture of at least four lipopeptides with seven amino acids incorporated and a fatty acid chain with 14 carbons, which makes this molecule similar to the biosurfactant of Bacillus pumilus, namely, pumilacidin. This is the first report on the biosurfactant production by B. safensis, encompassing the investigation of the metabolic pathway and chemical characterization of the biosurfactant molecule.

Surfactina: propriedades químicas, tecnológicas e funcionais para aplicações em alimentos

Surfactin, a lipopeptide produced by strains of Bacillus subtilis, has been proved to be a suitable biosurfactant in several applications. For many years, it has been investigated mainly for oil recovery and environmental usage. Its chemical, technological and functional characteristics turn surfactin into an attractive compound for several utilizations. In this review we emphasize some aspects of surfactin as a new food ingredient and its potential pharmaceutical and health applications.

Constituintes químicos e atividade antimicrobiana dos extratos de Dilodendron bipinnatum (sapindaceae)

The phytochemical investigation of ethanolic extracts from leaves, branches and stems of D. bipinnatum afforded the steroids β-sitosterol, stigmasterol, campesterol, sitostenone and sitosterol-3-O-β-D-glycopyranoside, along with two cycloartane triterpenes: cycloeucalenol and 24-methylenecycloartenol. The antimicrobial activity of the extracts was evaluated against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6623), Pseudomonas aeruginosa (ATCC 15442), Micrococcus luteus (ATCC 9341) and Candida albicans (ATCC 10231). The extracts of the leaves and branches showed moderate activity against Candida albicans. The extract of the branches was active against Micrococcus luteus. This is the first report on the phytochemical study of D. bipinnatum.

Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. citri

The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.

Secondary metabolites produced by Propionicimonas sp (ENT-18) induce histological abnormalities in the sclerotia of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a highly aggressive pathogen that causes great economic losses, especially in temperate climates. Several biological control agents are available, but actinobacteria have seldom been used to control this fungus. Our objective was to evaluate the efficiency and ultrastructural effects of the secondary metabolites produced by the ant-associated actinobacterium Propionicimonas sp. ENT-18 in controlling the sclerotia of S. sclerotiorum. We demonstrated total inhibition of sclerotia treated with 62.5 mu g/10 mu l of an ethyl acetate extract of compounds produced by ENT-18, and calculated an LC(50) of 1.69 mu g/sclerotia. Histological and ultrastructural analysis indicated that the cells of the treated sclerotia were severely damaged, suggesting direct action of the biomolecule(s) produced by the actinobacterium ENT-18 on the cell structure of the medullae and rind cell wall. This is the first report demonstrating a novel property of Propionicimonas sp.-antifungal activity against S. sclerotiorum.

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in `Valencia` oranges

Citrus black spot (CBS) caused by Guignardia citricarpa represents an important threat to citriculture in Brazil. Limited information is available regarding potential biological control agents and new alternative compounds that may provide protection of orange fruits against the disease. In this study, the effects of commercial products based on Bacillus thuringiensis var. kurstaki (Bt) bacterium, Bt pure isolates and Harpin protein (Messenger (R)) on the postharvest control of CBS, were evaluated in `Valencia` sweet orange fruits harvested for three consecutive years in a citrus grove. The fruits were sprayed with the following products: DiPel (R) WP (Bt, subspecies, kurstaki strain HD-1,16,000 International Units mg(-1), 32 g active ingredient kg(-1)) (1, 20 and 50 mg ml(-1)), Dimy Pel (R) WP (Bt, subspecies, kurstaki, strain HD-1, 17,600 IU mg(-1), 26 g active ingredient l(-1)) (2, 20 and 50 mg ml(-1)), Messenger (R) (3% harpin protein) (1 and 2 mg ml(-1)) and fungicide Tecto (R) Flowable SC (thiabendazole, 485 gl(-1)) (0.8g active ingredient l(-1)), besides the Bt isolates, Bt- HD-567, Bt- DiPel and Bt- Dimy (9 x 10(8) CFU ml(-1)). Ten days after treatment, the number of newly developed CBS lesions and pycnidia produced were evaluated using fifty fruits per treatment. The Dimy Pel (R) and Messenger (R) reduced the number of new developed CBS lesions on fruits in up to 67% and 62%, respectively. All applied treatments drastically decreased the number of pycnidia produced in the CBS lesions on orange fruits with 85% to 96% reductions compared to the untreated control. Volatile compounds produced by the isolates Bt- HD-567, Bt- Dimy and Bt- DiPel, reduced the number of lesions on treated fruits by 70%, 65% and 71% compared to the control, respectively. In addition, the survival of Bt isolates on orange fruit surfaces were evaluated by recovering and quantifying the number of CFU every seven days for up to 28 days. The declines in survival rates on orange fruit surfaces were drastic for the three strains of Bt in the first week. The CFU numbers of all applied isolates declined by 4 to 5 orders of magnitude after storage at room temperature for 28 days. In vitro assays revealed that the Bt isolates significantly reduced the mycelial growth of the pathogen, ranging from 32% to 51%, compared to the control, whereas no inhibitory effect was observed in the presence of Messenger (R). (C) 2010 Elsevier Ltd. All rights reserved.

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Sterilization by pure oxygen plasma and by oxygen-hydrogen peroxide plasma: An efficacy study

Plasma is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperatures. The use of different parameters for this method of sterilization and the corresponding results were analyzed in this study. A low-pressure inductive discharge was used to study the plasma sterilization processes. Oxygen and a mixture of oxygen and hydrogen peroxide were used as plasma source gases. The efficacy of the processes using different combinations of parameters such as plasma-generation method, type of gas, pressure, gas flow rate, temperature, power, and exposure time was evaluated. Two phases were developed for the processes, one using pure oxygen and the other a mixture of gases. Bacillus subtilis var. niger ATCC 9372 (Bacillus atrophaeus) spores inoculated on glass coverslips were used as biological indicators to evaluate the efficacy of the processes. All cycles were carried out in triplicate for different sublethal exposure times to calculate the D value by the enumeration method. The pour-plate technique was used to quantify the spores. D values of between 8 and 3 min were obtained. Best results were achieved at high power levels (350 and 40oW) using pure oxygen, showing that plasma sterilization is a promising alternative to other sterilization methods. (c) 2007 Elsevier B.V. All rights reserved.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.. Petiveria alliacea L. (Phytolaccacea) is an herbaceous plant of great importance in traditional medicine. This species have been widely used in several applications such as antirheumatic, anticarcinogenic, anti-flu, antitussive, analgesic, insecticidal, acaricidal, as well as bactericide and fungicide. Currently, the pathogenic microorganisms are acquiring resistence against the traditional antibiotics, and the search for new herbal antimicrobial agents has been intensified. The objective of this study was to evaluate the antifungal and antibacterial activity of several leaf crude extracts of P. alliacea against several strains of bacterias and yeasts namely Bacilus subtilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Candida parapsilosis, Candida kefyr and Candida albicans, using microdilution method. Promising results were observed for the 70% v/v ethanolic extract which presented minimum inhibitory concentration (MIC) from 250 to 760 mu g/mL for yeast. For the bacteria strains tested the MIC ranged between 240 to 3960 mu g/mL, depending of the extractive solution tested.

Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform(TM) was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform(TM) were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Characterization of lipopeptides from Paenibacillus sp (IIRAC30) suppressing Rhizoctonia solani

The main aim was to identify the active compound against Rhizoctonia solani produced by the cassava endophyte Paenibacillus sp. IIRAC-30. The compounds produced were extracted with ethyl acetate and purified by Sephadex column prior to analysis by Q-TOF mass spectrometry. A C(15)-lipopeptide with an estimated molecular weight of 1036 Da and homologues were identified. The lipopeptide had a cyclic structure, which was deduced by interpreting the ESI-MS/MS spectra of main protonated homologues containing 15:0 FA, and the amino acid composition was Glu-Leu-Leu-Val-Asp-Leu-Leu. Therefore, the lipopeptides produced by isolate IIRAC-30 was characterized as a surfactin series. Thus, the main mechanism used by Paenibacillus sp. IIRAC-30 to suppress R. solani was elucidated. Furthermore, because lipopeptides active against phytopathogens generally show low toxicity to humans and the environment, the positive findings presented here suggest that the isolate IIRAC-30 could be a possible candidate for biocontrol of R. solani.